qPCR guide SJ^Eurogentec Experience true partnership V 3 PREFACE Real-Time quantitative PCR, or qPCR in short, is heralded as the gold standard for accurate, sensitive and fast quantification of nucleic acid sequences. Indeed it is a wonderful technology, simple to perform and the risk for cross contamination is extremely low, as reaction tubes do not need to be opened. However, the simplicity of generating data is also the Achilles heel of qPCR. Nowadays, high quality reagents are available, and dedicated instruments do a perfect job. Hence, anyone that masters the skill of pipetting can generate beautiful sigmoidal amplification curves. What many investigators unfortunately fail to appreciate is quality control, which is essential throughout the entire qPCR workflow (from living cells, over extraction of nucleic acids, storage, various enzymatic steps such as DNase treatment, reverse transcription and PCR amplification, and finally data-analysis) in order to draw biologically meaningful conclusions. It is now well over 10 years after the conception of qPCR and there is still no consensus on good laboratory practice in general, and the level of quality control, experiment design and the data-analysis strategy in particular. As such, the life science literature is flawed with studies that are meaningless, inconclusive, or in the worst case erroneous (resulting in retraction of some papers). Fortunately, standards for data exchange and reporting guidelines are finding their way to the scientific community (see for example efforts made by the RDML consortium, http://www.rdml.org). When inspecting the scientific literature relating to qPCR, it is clearthat the number of publications over the years follows the same exponential rate as the PCR reaction itself. Currently, there is no plateau phase in sight; qPCR remains a growing market, especially in clinical diagnostics. In the research field, gene expression analysis is by far the most popular application, with double stranded DNA specific binding dyes and hydrolysis probes as the dominating detection chemistries (source: the Real-Time PCR Primer and Probe database, RTPrimerDB, http://medgen.ugent.be/rtprimerdb/). Coming from relatively low throughput qPCR systems a decade ago, 384-well instruments have now become more and more mainstream. "New kids" are now also on the block, implementing nanofluidic technologies, significantly increasing throughput and decreasing reaction volumes (down to nanoliter range), without the need for advanced liquid handling instruments. Following this promising path of ever increasing throughput, we might foresee a future in which we will simply PCR profile entire signalling networks or pathways, or even determine the expression level of almost every gene in an organism within only a few hours, with unprecedented sensitivity, accuracy and at low cost. Surely, it's a long road with many hidden obstacles, but witnessing the tremendous progress the science community have made in the last decade, the envisioned whole genome PCR profiling comes within reach. Jo Vandesompele Professor functional genomics and applied bio-informatics, Ghent University Co-founder and CEO, Biogazelle, the Real-Time PCR data analysis company EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide TABLE OF CONTENTS Introduction 6 Preliminary steps 7 Template preparation: DNA or RNA 7 One-step or two-step reaction? 8 Which type of primers for the Reverse Transcription? 9 Qualitative vs quantitative PCR technologies 10 What are qualitative PCR & quantitative PCR? 10 Quantitative PCR 11 Qualitative PCR 11 Detection methods 12 SYBR®Greenl 12 High Resolution Melting dyes (HRM dyes) 13 TaqMan® probes = Double-Dyes probes 13 LIMA® Double-Dye probes 15 Molecular Beacon probes 16 Scorpions® primers 18 Hybridization probes (also called FRET probes) 19 TaqMan® MGB® probes 20 MGB Eclipse® probes 20 Other technologies 21 When should I multiplex? 21 How do you get started in qPCR? 22 Thermocycler 22 qPCR reagents : formats and components? 24 Core kits or MasterMixes? 24 Role of core kits and MasterMixes components 24 Probe and primer quality 26 Manual coupling 26 Automatic coupling 26 Purification level 26 EUROGENTEC | www.eurogentec.com | info@eurogentec.com 5 Guidelines for primers and probes design 27 General rules 27 Design guidelines for SYBR® Green I assays 28 Design guidelines for Double-Dye Probe assays 29 Quenching principle 31 Fluorophore and quencher: definitions 31 Different ways of quenching 31 FRET quenching 32 Optimal flurophore quencher combination 32 Dye-thermocyclers compatibility table 34 Double-Dye Oligonucleotides in single-color detection 34 Double-Dye Oligonucleotides for multiplexing detection 34 Example of a classical assay 36 Some tips and tricks before starting 36 Classical protocol and thermal profile 37 Useful definitions 38 Normalization and quantification methods 39 Absolute quantification 41 Relative quantification 41 Evaluation of your qPCR assay 43 Controls 43 Negative controls 43 Positive controls 44 Commonly used control kits 45 Singleplex or multiplex assay for the controls? 45 Role of the standard curve and evaluation of its parameters 46 PCR efficiency 46 R square (R2) 47 Sensitivity 47 Reproductibility 47 Specificity of a SYBR® Green I assay 47 Optimization tips 49 In general 49 For probe multiplexing assay/qualitative qPCR 50 Troubleshooting guide 51 Frequently Asked Questions 53 Eurogentec products 55 References 64 EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide This guide is dedicated for both beginners and experts in qPCR. It explains both the basics of qPCR and gives useful tips for troubleshooting. Real-Time qPCR is a technique that involves a high number of parameters, all of them having to be optimized to get the highest quality assay, and all of which are specific to one assay only. There is consequently no "magic formula". However, this booklet can provide tools to help you, from your first contact with qPCR to the optimization steps. If you are an expert, just go straight to the last pages of this booklet. We hope that by working through the pages, we will help you enjoy this technique and reach the best results for your research! INTRODUCTION The Polymerase Chain Reaction (PCR) has been invented in 1983 by Kary Mullis (Nobel Price in 1993), (Mullis K. et Fobona R, 1987). Three years after its invention, there was an incredible expansion of its use thanks to the commercialization of the Taq polymerase, a polymerase that resists high temperatures. In 1991, the first hydrolysis probe was used in combination with the technique. In 1992, the technique was again improved by the use of Ethidium Bromide (EtBr), thanks to the fluorescence that results from the binding to duplex DNA. The kinetics of fluorescence accumulation during thermocycling was directly related to the starting number of DNA copies. This was the starting point of Real-Time qPCR. Today, 25 years after Mullis's discovery, both PCR and qPCR are widely used technologies. The principle, and aim, of the PCR technology is to specifically increase a target from an undetectable amount of starting material. In classical PCR, at the end of the amplification, the product can be run on a gel for detection of this specific product. In Real-Time PCR, this step can be avoided since the technology combines the DNA amplification with the immediate detection of the products in a single tube. The homogeneous format is highly beneficial as it removes the significant contamination risk caused by opening tubes for post-PCR manipulation. It is also less time consuming than gel based analysis and can give a quantitative result (Kubista M.ef a/., 2006). Current detection methods are based on changes in fluorescence, which are proportional to the increase of target. Fluorescence is monitored during each PCR cycle providing an amplification plot, allowing the user to follow the reaction in real time. (Figure 1). Delta Rn vs Cycle 7000 6000 / 5000 ) 1 1 1 / '/ 'l 1 4000 1 I \ • ll 'I 'l / 1 3000 ' 1 1 1 1 1 2000 / l 1 t / \ / / 1000 / / 0 Cycle Number Figure 1. Amplification plot of 10 x serial diluted cDNA, using SYBR® Green detection method, on an ABI 7500 thermocycler. EUROGENTEC | www.eurogentec.com | info@eurogentec.com PRELIMINARY STEPS Variability in qPCR is often related to steps upstream to the qPCR step itself. Sample extraction, quantity of sample, or efficiency of the reverse transcription (RT) are some of the many parameters that can influence the results of your qPCR assay. That's why it is important to also consider the quality of these steps prior to performing your qPCR assay (Fleige S. and Pfaffl MW, 2006). Template preparation: DNA or RNA DNA and RNA can be extracted with Trizol. This includes many steps and a careful wash of the sample, hence we recommend the use of an appropriate commercially available kit. Many column-based kits, which will contain all the required reagents for the full extraction / purification procedure are available. These kits will also outline general guidelines, such as the storage conditions and shelf life of the extracted RNA or DNA. However these guidelines may vary between kits due to the different composition of buffers. It is also recommended that the buffers supplied with each kit are used according to the manufacturer protocol. The final product should be cleaned and free from any residual buffers such as EDTA or buffers containing solvents. Otherwise, these residual products may inhibit the action of the Taq polymerase for the PCRstep, or could modify the salt concentration of the buffer. This is usually not a problem when using spin column kits instead of manual extraction techniques. It is worth bearing in mind, that this first step is nearly the most important of your RT-qPCR assay, as the quality of the extraction will influence the quality of your detection and quantification. It's mostly important to ensure the reproducibility of your extraction steps, if you wish to compare biological samples. The main problems that can occur, are extraction of inhibitors together with the nucleic acids or degradation of the sample. Inhibitors are mainly found in blood sample or environmental samples. For example, humic acid can be a strong inhibitor in samples extracted from soil. As a general rule, it is always better to extract DNA/RNA from fresh sample and to store it at -80 °C. EUROGENTEC | www.eurogentec.com | info@eurogentec.com qPCR guide One-step or two-step reaction? If your starting material is total RNA, you have to perform the Reverse Transcription (RT) step before your qPCR assay. This can be done in One-Step or Two-Steps. Both methods have pros and cons you might consider to choose the best one for your application. In both cases, the RT reaction should be set up in a clean environment to avoid contamination. We also recommend working with RNase free plastics. The tubes containing the reaction should be maintained on ice during the set up of the reaction. This will prevent an early reaction, generated before the incubation period. The RT step lasts 30 minutes around 50 °C. However, if random primers or oligo d(T) are used (two-step reaction), an initial 10 min-step at 25 °C is necessary to maximize the annealing efficiency, as the short oligo d(T) have a lower Tm. The next paragraph explains what kind of primers can be used under which circumstancies. The cDNA generated should be stored at -80 °C. The use of a separate RT step is recommended when the reaction is performed with a limiting amount of starting material. One-Step qRT-PCR Two-step qRT-PCR Description RT and qPCR reactions performed in the same tube Optimized working buffer for both the RT and PCR enzymes RT and qPCR reactions performed in separate tubes Pros Save pipetting steps No contamination between RT and qPCR steps More efficient because random primers and oligo d(T) can be used Possibility to stock cDNA to quantify several targets Lower background in SYBRB assays Best option for High-Throughput Screening (less time consuming than 2-step reactions) More flexible (separate optimization possible for the two reactions) Cons No possibility to use UNG carry-over prevention Usually less sensitve than a Two-Steps qRT-PCR assay RNase inhibitors that can influence the PCR reaction after the RT Higher background when performing a SYBRB assay Table 1: Pros and cons of One-Step qRT-PCR versus Two-Step qRT-PCR EUROGENTEC | www.eurogentec.com | info@eurogentec.com 9 Which type of primers for the Reverse Transcription? When performing a RT, it is possible to use three different types of primers: • short random primers, which bind anywhere in the genome and allow the reverse transcriptase to fill up the gaps, lead to higher yields • oligo d(T) bind to the poly-A tail of the RNA and then only transcribe RNA. This will avoid contamination by genomic DNA. As the poly-A tail is located at the extremity of the gene, it will also lead to more full transcripts • specific primers, which bind to the gene of interest The combination of oligo d(T) primers and random primers will give the highest yields and the longest transcripts, whereas specific primers transcribe only specific RNA but reduce the yield. With a One-Step qRT-PCR reaction, using specific primers is the only option, as it should be avoided that oligo d(T) primers and random nonamers participate in the PCR step, giving many aspecific products. The RT step is performed at 40-50 °C and the specific PCR primers are designed for the PCR step at 60 °C, leading to partial annealing of the primers during the RT step. In a Two-Steps qRT-PCR kit, oligo d(T) primers or/and random nonamers are used in the RT step, and specific primers in the PCR step, leading to specific cDNA. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide QUALITATIVE vs. QUANTITATIVE PCR What are qualitative PCR & quantitative PCR? Firstly it is important to review the principles of PCR, before making the distinction between qualitative and quantitative PCR. The action of the Taq polymerase allows extension of short single-stranded synthetic oligonucleotides (i.e. primers) during repeated cycles of heat de-naturation, primer annealing and primer extension. The primers are designed to specifically bind the extremities of the DNA fragment to be amplified. The Taq polymerase uses the target DNA added to the reaction as a template for primer extension. At each cycle, more DNA is synthesized, creating more template DNA. The reaction proceeds in an exponential manner, doubling the amount of target during each cycle, until one of the reagents becomes limiting and the reaction reaches a plateau. ►So + 1 1 1 1 1 I i I i Figure 2. The Polymerase Chain Reaction. (K. Mullis and F. Fabona, 1987). The red and pink lines represent the primers that the polymerase extends from. The blue and black lines represent the single-stranded DNA template produced from de-naturation of the double-stranded DNA template. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 11 Quantitative PCR In quantitative qPCR, a specific or non-specific detection chemistry allows the quantification of the amplified product. The amount detected at a certain point of the run is directly related to the initial amount of target in the sample. The most common applications of quantitative PCR are gene expression analysis, pathogen detection/quantification and microRNA quantification (Schmittgen TD ef a/.,2008). For example, effects of different treatments, on the level of mRNA transcription can be measured. Quantitative PCR software uses the exponential phase of PCR for quantification. PCR is initially an exponential process but eventually reaches a plateau phase, when one of the reagents becomes limited. Reactions can plateau at different levels even if they have the same starting concentration of target. During the exponential phase, the amount of target is assumed to be doubling every cycle and no bias is expected due to limiting reagents. Analysis takes the Ct (cycle number) value, at the point when the signal is detected above the background and the amplification is in exponential phase. The more abundant the template sample, the quicker this point is reached, thus giving earlier Ct values. Differences in Ct then have to be correlated to some other quantitative values to make them meaningful (cf. p. 39). Qualitative PCR In qualitative qPCR, the goal is to detect the presence or absence of a certain sequence. It could be for vims sub-typing and bacterial species identification for example. It can also be used for allelic discrimination between wild type and mutant, between different SNPs (Single Nucleotide Polymorphisms) or between different splicing forms. In this case, different fluorophores can be used for the two alleles, and the ratio of the fluorophores signals correlates to the related amount of one form compared to the other one. Specific detection methods such as Double-Dye probe systems are more often used for these applications, and probes can be used to detect single base mutations or small deletions. Recently, a new technique called HRM (High Resolution Melting) emerged, allowing gene scanning and SNP genotyping without using probes (cf. p. 13). • — - - -e hi h — - j ■ - i -- J * -- • -1 TP fft- X- r t i 11 Allele X Fluorescent Signal Figure 3. Allelic discrimination using two Double-Dyes labelled with different fluorophore (ncvs.org). EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Detection methods In order to detect and measure the amount of target in the sample, a measurable signal has to be generated, which is proportional to the amount of amplified product. All current detection systems use fluorescent technologies. Some of them are non-specific techniques, and consequently only allow the detection of one target at a time. Alternatively, specific detection chemistries can distinguish between nonspecific amplification and target amplification. These specific techniques can be used to multiplex the assay, i.e. detecting several different targets in the same assay. SYBR® Green I SYBR® Green I is the most commonly used dye for non-specific detection. It is a double-stranded DNA intercalating dye, that fluoresces once bound to the DNA. A pair of specific primers is required to amplify the target with this chemistry. The amount of dye incorporated is proportional to the amount of generated target. The dye emits at 520 nm and fluorescence emitted can be detected and related to the amount of target. The inconvenience of this technique is that the SYBR® Green I will bind to any amplified dsDNA. Consequently, primer dimers or unspecific products introduce a bias in the quantification. However, it is still possible to check for the specificity of the system by running a meltcurve at the end of the PCR run (cf. p. 47). The principle is that every product has a different dissociation temperature, depending of the size and base contents, so it is still possible to check the number of products amplified. A valid SYBR® assay - primer pair - should produce a unique, well defined peak on the meltcurve. For these reasons, SYBR® Green I is rarely used for qualitative PCR. However, SYBR® Green I is often used as the first step to optimize a specific detection system assay, to check the specificity of the primers and validate the design. SYBR® Green I advantages • Low cost assay • Easy design and set up SYBR® Green I disadvantages • Non specific system • Not adapted to multiplex • Non suitable for qualitative qPCR EUROGENTEC | www.eurogentec.com | info@eurogentec.com 13 High Resolution Melting dyes (HRM dyes) High Resolution Meltcurve analysis is a newly emerging technology, which characterizes nucleic acid samples based on their dissociation behaviour. It combines the principle of intercalating dyes, meltcurve analyses and the application of specific statistical analyses. HRM uses the fundamental property of the separation of the two strands of DNA with heat (melting), and the monitoring of this melting with a fluorescent dye. On the contrary of SYBR Green, HRM dyes do not inhibit PCR at high concentration. The dye can consequently saturate the amplified target dsDNA and fluoresces. Melting temperature of a dsDNA target depends on GC content, length, and sequence. Due to the high sensitivity of HRM dyes, even a single base change will induce differences in the melting curve, and consequently in fluorescence (Erali M. ei a/., 2008). Main applications of HRM include gene scanning (search for the presence of unknown variations in PCR amplicons), SNP genotyping, DNA methylation analysis, DNA mapping, DNA fingerprinting. This emerging method is less expensive and as precise than probe-based methods. Only a few thermocyclers on the market currently allow the use of this technology, among them the Roche LightCycler®480, the Corbett Life Science Rotor-Gene™ 6000, and the ABI Prism® 7500. The main HRM dyes available are EvaGreen, LCGreen®, SYTO8 9 and BEBO. TaqMan® probes = Double-Dye probes TaqMan® probes, also called Double-Dye Oligonucleotides, Double-Dye Probes, or Dual-Labelled probes, are the most widely used type of probes and are often the method of choice for scientists who have just started using Real-Time PCR. They were developed by Roche (Basel, Switzerland) and ABI (Foster City, USA) from an assay that originally used a radio-labelled probe (Holland ef al. 1991), which consisted of a single-stranded probe sequence that was complementary to one of the strands of the amplicon. A fluorophore is attached to the 5' end of the probe and a quencher to the 3' end. The fluorophore is excited by the machine and passes its energy, via FRET (Fluorescence Resonance Energy Transfer) to the quencher. Traditionally the FRET pair has been FAM as the fluorophore and TAMRA as the quencher. In a well designed probe (cf. Taqman® design rules on p. 30), FAM does not fluoresce as it passes its energy onto TAMRA. As TAMRA fluorescence is detected at a different wavelength to FAM, the background level of FAM is low. The probe binds to the amplicon during each annealing step of the PCR. When the Taq polymerase extends from the primer which is bound to the amplicon, it displaces the 5' end of the probe, which is then degraded by the 5'-3' exonuclease activity of the Taq polymerase. Cleavage continues until the remaining probe melts off the amplicon (cf. fig. 4). This process releases the fluorophore and quencher into solution, spatially separating them (compared to when they were held together by the probe). This leads to an irreversible increase in fluorescence from the FAM and a decrease in the TAMRA. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide |-|.:».nJ I..!.. 1 1 f F V-T --v /__, Figure 4. TaqMan" probes mode of action TaqMan® probes can be used for both quantification and mutation detection, and most designs appear to work well. They are convenient either for allelic discrimination or expression profiling and are usually easy to design, easy to use in a standard protocol and need minimal optimization. For mutation detection, the probe is designed to hybridize over the mutation site and can be made specific enough to detect single base differences. To obtain robust allelic data, it is vital that a different probe is used for each different mutation, otherwise negative results with one probe, (caused by failed PCR reactions), can be scored as an absence of a particular allele. Ideally these probes would be multiplexed to make the assays faster and cheaper. Double-Dye probes advantages • Widely used, several modifications possible • Specific system • Multiplex capabilities Double-Dye probes disadvantages • More expensive than SYBR® Green I, but the lowest cost specific system available EUROGENTEC | www.eurogentec.com | info@eurogentec.com 15 LNA® Double-Dye probes LNA® (Locked Nucleic Acid) was developed by Exiqon® (Vedbaek, Denmark). LNA® changes the conformation of the helix and increases the stability of the duplex. The integration of LNA® bases into Double-Dye Oligonucleotide probes, opens up great opportunities to improve techniques requiring high affinity probes as specific as possible, like SNP detection, expression profiling and in situ hybridization. LNA® is a bicyclic RNA analogue, in which the ribose moiety in the sugar-phosphate backbone is structurally constrained by a methylene bridge between the 2'-oxygen and the 4'-carbon atoms. Figure 5. LNA backbone structure The integration of LNA® bases into probes changes the conformation of the double helix from the B to A type (Ivanova A. ef a/., 2007). Figure 6. B helix and A helix containing LNA bases LNA® conformation allows a much better stacking and therefore a higher stability. By increasing the stability of the duplex, the integration of LNA® monomers into the oligonucleotide sequence allows an increase of the melting Temperature (Tm) of the duplex. It is therefore possible to reduce the size of the probe, which increases the specificity of the probe and helps designing it (Karkare S. ef a/., 2006). LNA® advantages • Increases thermal stability towards complementary DNA and RNA • Allows use of shorter probes • High specificity and reproducibility • Simplifies multiplex assays by adjusting Tm values of primers and probes EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Molecular Beacon probes Molecular Beacons are probes that contain a stem-loop structure, with a fluorophore and a quencher at their 5' and 3' ends, respectively. The stem is usually 6 bases long, should mainly consist of C's and G's, and holds the probe in the hairpin configuration (Li Y. ef a/., 2008). The 'stem' sequence keeps the fluorophore and the quencher in close vicinity, but only in the absence of a sequence complementary to the 'loop' sequence. As long as the fluorophore and the quencher are in close proximity, the quencher absorbs any photons emitted by the fluorophore. This phenomenon is called collisional (or proximal) quenching. In the presence of a complementary sequence, the Beacon unfolds and hybridizes to the target, the fluorophore is then displaced from the quencher, so that it can no longer absorb the photons emitted by the fluorophore, and the probe starts to fluoresce. The amount of signal is proportional to the amount of target sequence, and is measured in real time to allow quantification of the amount of target sequence (Takacs T. ef a/., 2008). The increase in fluorescence that occurs is reversible, (unlike TaqMan® probes), as there is no cleavage of the probe, that can close back into the hairpin structure at low temperature. The stem structure adds specificity to this type of probe, because the hybrid formed between the probe and target has to be stronger than the intramolecular stem association. Good design of Molecular Beacons can give good results, however the signal can be poor, as no physical separation of fluorophore from quencher occurs. -i > J 20 000 —| 1 5 000 - ^""■"V. Beacon-target hybrid £ 1 0 000 -5 000 - C ) a M Beacon without target o -1 5 1 35 1 1 55 75 Temperature (°C) Figure 7. Melt curve of a Molecular Beacon with and without a synthetic complement as a target. Annotations show the configuration of the beacon and target at different temperatures. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 17 Two improved forms of Molecular Beacons exist: • Wavelength-Shifting Molecular Beacons Wavelength-Shifting Molecular Beacons are brighter than standard Molecular Beacons due to an enhanced fluorescence intensity of the emitter fluorophore. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a non-fluorescent (dark) quencher. In the absence of complementary nucleic acid targets, the probes are non-fluorescent, whereas in the presence of targets, they fluoresce, not in the emission range of the harvester fluorophore, that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by FRET, which only takes place in probes that are bound to the targets. Wavelength-Shifting Molecular Beacons are substantially brighter than conventional Molecular Beacons that cannot efficiently absorb energy from the available monochromatic light source (Tyagi S. ef a/., 2000). • 2' O-Methyl RNA Molecular Beacons To detect the various RNA classes in living cells, several approaches have been developed. One of these is based on the use of 2'-0-Methyl RNA Molecular Beacon probes for the detection of small nuclear RNAs. 2'-0-methyl RNA probes are considered to perform better than DNA oligonucleotides because they are not only nuclease resistant, but also possess a higher affinity, increased specificity, faster hybridization kinetics, and a superior ability to bind to structured targets compared to DNA oligonucleotides. Recently Molecular Beacons were introduced for RNA detection in living cells. The rationale for using Molecular Beacons to detect RNAs in living cells was to improve signal-to-noise ratios by eliminating fluorescence signals derived from non-hybridized probe sequences. It appears that linear 2'-0-Methyl RNA probes are usually more suitable for specific detection of these RNAs, representing different classes of RNA, in the nuclei of living cells. Molecular Beacons result in images with improved signal to noise ratios, thereby leading to better detection sensitivity. Molecular Beacons • Reversible phenomenon (melting curve possible) • Increased discriminatory competence due to competition intra-inter target EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Scorpions® primers Scorpions® primers are suitable for both quantitative Real-Time PCR and genotyping/end-point analysis of specific DNA targets. They are PCR primers with a "stem-loop" tail consisting of a specific probe sequence, a fluorophore and a quencher. The "stem-loop" tail is separated from the PCR primer sequence by a "PCR blocker", a chemical modification that prevents the Taq polymerase from copying the stem loop sequence of the Scorpions® primer. Such read-through would lead to non-specific opening of the loop, causing a non-specific fluorescent signal. The hairpin loop is linked to the 5' end of a primer via a PCR blocker. After extension of the primer during PCR amplification, the specific probe sequence is able to bind to its complement within the same strand of DNA. This hybridization event opens the hairpin loop so that fluorescence is no longer quenched and an increase in signal is observed. Figure 8. Scorpions" unimolecular mode of action. Progression through a PCR reaction. Unimolecular probing is kinetically favorable and highly efficient. Covalent attachment of the probe to the target amplicon ensures that each probe has a target in the near vicinity. Enzymatic cleavage is not required, thereby reducing the time needed for signaling compared to TaqMan® probes, which must bind and be cleaved before an increase in fluorescence is observed. Scorpions® primers have successfully been used for mutation detection and quantification (Solinas A. etal., 2001), having the specificity and melt curve analysis of the Molecular Beacons, with additional speed and efficiency. However, constraints associated with Scorpions® design is probably the reason why this type of probe is not as successful as it should be. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 19 There are three types of Scorpions® primers : • Standard Scorpions®, which consist of a bi-labelled probe with a fluorescent dye at the 5' end and an internal non-fluorescent quencher. • FRET Scorpions®, for use on a LightCycler® system. As the capillary system will only excite at 470 nm (FAM absorption wavelength) it is necessary to incorporate a FAM within the stem. A ROX is placed at the 5'end of the Scorpions® primer, FAM is excited and passes its energy onto the ROX. • Duplex Scorpions® have also been developed to give much better signal intensity than the normal Scorpions® format. In Standard Scorpions® the quencher and fluorophore remain within the same strand of DNA and some quenching can occur even in the open form. In the Duplex Scorpions® the quencher is on a different oligonucleotide and physical separation between the quencher and fluorophore is greatly increased, reducing the quenching when the probe is bound to the target. Advantages of Scorpions® primers vs Taqman® probes • Stronger signal and lower background • Improved SNP applications • Compatible with any dye, convenient for extended multiplexing assays • Primer and probe on one molecule, kinetically favorable, fast cycling Roche has developed hybridization probes (Caplin ef al. 1999) for use with their LightCycler®. Two probes are designed to bind adjacent to one another on the amplicon. One has a 3' label of FAM, whilst the other has a 5' LC dye, LC red 640 or 705. When the probes are not bound to the target sequence, the fluorescent signal from the reporter dye is not detected. However, when the probes hybridize to the target sequence during the PCR annealing step, the close proximity of the two fluorophores allows energy transfer from the donor to the acceptor dye, resulting in a fluorescent signal that is detected. conditions Hybridization probes (also called FRET probes) 5 ■ Figure 9. FRET probe principle EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide TaqMan® MGB® probes TaqMan® MGB® probes have been developed by Epoch Biosciences (Bothell, USA) and Applied Biosystems (Foster City, USA). They bind to the minor groove of the DNA helix with strong specificity and affinity. When the TaqMan® MGB® probe is complemented with DNA, it forms a very stable duplex with DNA. The probe carries the MGB® moiety at the 3' end. The MGB strongly increases the probe Tm , allowing shorter, hence more specific designs. The probe performs particularly well with A / T rich regions, and is very successful for SNP detection (Walburger ef a/., 2001). It can also be a good alternative when trying to design a probe which should be located in the splice junction (for which conventional probes are hard to design). Smaller probes can be designed with Tm as 65-67 °C, which gives a better discrimination (the probe is more specific for single mismatch). A good alternative to MGB probes are LNA® probes where the increase in Tm induced by the addition of LNA® bases is specific, contrary to the MGB moeity (cf. p. 15). During the primer extension step, the hybridized probe is cleaved by the 5' exonuclease activity of Taq polymerase and an increase in fluorescence is seen. Fluorescence of the cleaved probe during PCR is monitored in Real-Time by the thermocycler. 1, MJ' r,n»ir ■ MSB' t-r*. i © t Figure 10. TaqMan® MGB® probe principle MGB Eclipse® probes MGB Eclipse® probes also known as QuantiProbes, have originally been developed by Epoch Biosciences (Bothell, USA). MGB Eclipse® probes carry a minor groove binder moiety that allows the use of short probes for very high specificity. These are short linear probes that have a minor groove binder and a quencher on the 5' end and a fluorophore on the 3'end. This is the opposite orientation to TaqMan® MGB® probes and it is thought that the minor groove binder prevents the exonuclease activity of the Taq polymerase from cleaving the probe. The quencher is a Non Fluorescent Quencher also known as Eclipse Dark Quencher. Quenching occurs when the random coiling of the probe in the free form brings the quencher and the fluorophore close to another. The probe is straightened out when bound to its target and quenching is decreased, leading to an increase in fluorescent signal. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 21 Other technologies The technologies that have been discussed above are the most widely used today, but numerous other technologies have occurred in publications, or are available on the market, such as: Resonsense probes, Light-up probes, HyBeacon® probes, LUX primers, Yin-yang probes, or Amplifluor®. You can contact us for more information on any of them. When should I multiplex? We have previously seen that some chemistries are more adapted to multiplexing than others. Theoretically, multiplex assay can be performed also with a non-specific system such as SYBR® Green I assay. The most common use of multiplex reaction is for qualitative PCR assay. In a quantitative assay, it mainly allows to include a control gene in the same well as the target (cf. p. 39, Normalization and Quantification methods). Multiplexing is sometimes a necessity (SNP detection), but multiplex assays can also be set up for practical reasons, to save time and reagents (Ishii T. ef a/., 2007). When is it worth multiplexing? When is it better to singleplex? • For SNP/mutation analysis: for bi-allelic discrimination, one specific probe is designed for each allele. Both of them have a 5' end fluorophore and a quencher at the 3' end and duplex assay is necessary. • When targeting two genes with equal expression levels with PCR reactions of similar amplification efficiency, multiplex Real-Time PCR can be performed without any doubt. • When analyzing two genes with different expression levels we recommend separating amplification of targets. • If you quantify lots of different targets on few samples, it is not necessary to multiplex as you won't save time. Optimizing a multiplex reaction is time-consuming, so it is worth doing it if you plan to work on a large cohort of samples, and always on the same targets. • If you have strict requirement for your design (cross linked species...), avoid multiplexing, as you can observe annealing between probes or primers, if your design is not optimal. • However if you manage to have a good PCR efficiency for your singleplex assays, if you don't see any complementarities between your probe and primers, and if you routinely work with the same genes, then it is worth optimizing your multiplex reaction. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide HOW DO YOU GET STARTED IN qPCR? Thermocycler It seems logical, but you need to get a good thermocycler before you start your qPCR assay. You will most certainly start yourqPCR project with the thermocycler available in your lab, or the lab nearby. The majority of the thermocyclers on the market now offer similar characteristics. The table shows the main thermocyclers on the market, and can be considered as a starting point in your decision making process. The following characteristics should always be taken into consideration: • Format: glass capillaries, plastics tubes, 96-well plates or 384-wells plates. Consider this aspect depending on your requirements, and also based on the reagent availability. A plate format is ideal if you have a high throughput requirement. Glass capillaries can reduce the choice of reagents which you may want to use. • Number of detection channels: if you want to multiplex, choose a thermocycler with a large choice of dyes, in order to have the best combination for your assay. You can find on p. 35 the table showing available dye combinations, based on the type of thermocycler. • Software analysis: Consider the simplicity of the software, the format to export data, statistical analysis performed etc. In addition to qPCR analysis, you can also perform High Resolution Meltcurve analysis with some thermocyclers. • Time of run: the newest thermocyclers enable a very short time of run (30-45 minutes) compared to some of the older ones which take 2 hours. • Price and flexibility of the offer. Some suppliers try to tie up the machine with their corresponding disposable and reagents. EUROGENTEC | www.eurogentec.com | info@eurogentec.com m c o a m m O S1 CD 5 Thermocycler Supplier Format Number of channels Optical Excitation system Notes More information on: GeneAmp® SDS 5700 Applied Biosystems 96-well plate 1 Halogen lamp Sales discontinued http://www.appliedbiosystems.com/ ABI Prism* SDS 7000 Applied Biosystems 96-well plate 4 Tungsten-halogen lamp Sales discontinued http://www.appliedbiosystems.com/ ABI Prism* SDS 7700 Applied Biosystems 96-well plate 4 Laser Sales discontinued http://www.appliedbiosystems.com/ ABI Prism* SDS 7900 HT Applied Biosystems 96-well plate or 384-well plate 4 Laser Robotic plate loading system for high throughput use Normal or FAST block http://www.appliedbiosystems.com/ ABI Prism* SDS 7300 Applied Biosystems 96-well plate 4 Tungsten-halogen lamp http://www.appliedbiosystems.com/ ABI Prism* SDS 7500 Applied Biosystems 96-well plate 5 Tungsten-halogen lamp Normal or FAST block http://www.appliedbiosystems.com/ StepOne* / StepOnePlus* Applied Biosystems 48-well /96-well 3/4 LEDs http://www.appliedbiosystems.com/ iCycler iQ* BioRad 96-well plate 4 Halogen lamp Sales discontinued http://www.bio-rad.com/ MyiQ* BioRad 96-well plate 1 Tungsten- halogen lamp Singleplex FAM/SYBR http://www.bio-rad.com/ iQ5* BioRad 96-well plate 5 Tungsten-halogen lamp http://www.bio-rad.com/ CFX 96* BioRad 96-well plate 5 LEDs PCR cycler converted into Real-Time cycler http://www.bio-rad.com/ MiniOpticon® BioRad 48-well plate 2 LEDs http://www.bio-rad.com/ DNA Engine Opticon* 1 BioRad 96-well plate 1 LEDs Sales discontinued http://www.bio-rad.com/ DNA Engine Opticon* 2 BioRad 96-well plate 2 LEDs http://www.bio-rad.com/ Chromo 4* BioRad 96-well plate 4 LEDs http://www.bio-rad.com/ Mx3000P* Stratagene 96-well plate 4 Quartz tungsten-halogen lamp Customizable filters http://www.stratagene.com/ MX3005P* Stratagene 96-well plate 5 Quartz tungsten-halogen lamp Customizable filters http://www.stratagene.com/ Mx4000* Stratagene 96-well plate 4 Quartz tungsten-halogen lamp Sales discontinued http://www.stratagene.com/ Mastercycler ®ep realplex2 Eppendorf 96-well plate 2 LEDs http://www.eppendorf.com/ Mastercycler ®ep realplex4 Eppendorf 96-well plate 4 LEDs http://www.eppendorf.com/ LightCycler* 1.5 Roche 32 glass capillaries 3 LEDs https://www.roche-applied-science.com/ LightCycler® 2 Roche 32 glass capillaries 6 LEDs https://www.roche-applied-science.com/ LightCycler 480® Roche 96-well or 384-well plate 6 Xenon lamp Optional HRM module https://www.roche-applied-science.com/ Smartcycler® Cepheid Innovation Up to 96 plastic tubes. 4 LEDs Independent block of 16 wells. Up to 6 blocks can be combined. http://www.cepheid.com/ Rotor-Gene™ 2000 / 3000 Corbett Life Science 16/32 or 72 plastics tubes 2/4 LEDs Sales discontinued http://www.corbettlifescience.com Rotor-Gene™ 6000 Corbett Life Science 6 LEDs Includes HRM module http://www.corbettlifescience.com cf. the dye-thermocycler compatibility table on p. 35 qPCR guide qPCR reagents : formats and components? When choosing a reagent for your qPCR assay, consider the format and the composition, in order to use one which is adapted to your assay, your machine, and your way of working. Core kits or MasterMixes? A Core kit, is a kit that contains all components in separate tubes, so you have to mix them yourself. It gives a maximum of flexibility, as you can optimize the concentration of each component of your assay. A qPCR Core kit contains individual tubes of reaction buffer, dNTP/dUTP mix, MgCI2, Taq and eventually SYBR® Green. On the contrary, a MasterMix is a ready-to-use reagent in which all the components are already mixed in an optimized way. Additional stabilizers are added to enable long-term storage. The concentration of components is perfectly adapted to most of the assays. Consequently, the 2 x mix guarantees a high reproducibility, ease of use, and time saving. Role of Core kits and MasterMixes components Slight component changes can significantly impact your assay. It is therefore important to consider them individually, understanding the role of each of them. Taq Polymerase A HotStart Taq polymerase is inactive at low temperatures (room temperature). Heating at 95 °C for several - usually 5 to 10 - minutes activates the enzyme, and the amplification can begin once the primers are annealed. The enzyme is not active until the entire DNA is denatured. Two major HotStart modifications exist, the antibody-blocked Taq and the chemically-blocked Taq. The antibody-blocked Taq is inactive because it is bound to a thermolabile inhibitor that is denatured during the initial step of PCR. The chemically-blocked Taq provides one clear advantage over the antibody-blocked Taq, as it is completely inactive at 60 °C, (the hybridization temperature of primers), thus preventing the formation of nonspecific amplification and reducing primer dimer formation. dNTPs/dUTPs Some kits contain a blend of dNTPs and dUTPs, other ones contain only dNTPs. Using only dNTPs increases the sensitivity, the reason being that the Taq incorporates more easily dNTPs than dUTPs. However, using a mix containing dUTPs brings security to the assay, in case of contamination from a previous PCR product. Thanks to the UNG activity in association with incorporated dUTPs, this contamination can be eliminated. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 25 UNG The Uracil-N-Glycosylase is an enzyme that hydrolyses all single-stranded and double-stranded DNA containing dUTPs. Consequently, if all PCR amplifications are performed in the presence of a dNTPs/dUTPs blend, by carrying a UNG step before every run it is possible to get rid of any previous PCR product. ROX Some thermocyclers require MasterMix containing ROX dye for normalization. This is the case for the ABI and Eppendorf machines, and optional on the Stratagene machines. If you work with such machines, it is easier to work with the ROX dye already incorporated in the MasterMix rather than adding it manually. It guarantees a higher level of reproducibility and homogeneity of your assays. Fluorescein For iCycler iQ®, My iQ® and iQ5 machines (BioRad thermocyclers), the normalization method for SYBR® Green assay uses Fluorescein to create a "virtual background". As in the case for the ROX, it is better and easier to use a MasterMix that contains pre-diluted Fluorescein, guaranteeing higher reproducibility and homogeneity of your assays. MgCI2 MgCI2 is necessary for the Reverse Transcriptase and the Taq activity. MgCI2 concentration in MasterMixes is optimized according to the amount of Taq and also the buffer composition. However, it may be necessary sometimes to add MgCI2 and most MasterMixes include an additional tube of MgCI2. Inert colored dye Some buffers also include an inert colored dye, to enable visualization of the buffer when loading in the wells. This colored dye has no effect on the sensitivity of the assay and is a convenient working tool. Note that such mixes, in combination with white plastic plates, provide better levels of fluorescence and a really easy way of working. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Probe and primer quality We really cannot insist enough, about the importance of this parameter. Your design is actually the most variable parameter, especially in the case of SYBR® Green assays. It is not uncommon to observe undetectable targets, high backgrounds or bad PCR efficiency, due only to the poor design of the primers and probes. The next chapter details more precisely, the rules for probe and primer design. However, consider also the quality and the purification level of your primers and probes, as this can also influence your results. The classical way of synthesizing oligonucleotides, is the standard Beta-cyanoethyl synthesis cycle that includes: deblocking the first nucleotide, coupling of the second one, capping of any previous nucleotide that has not been coupled, oxidation to stabilize the growing chain, deblocking, then a new cycle starts. At the end of the oligonucleotide synthesis, the crude product is cleaved from the solid support (CPG or polystyrene beads). Concerning probe coupling, there are different methods by which a dye can be incorporated into a probe. The choice of the coupling method used depends on the structure of the dye and of the length of the probe. Manual coupling Manual coupling can be done via amine or thiol groups, using activated dyes such as Texas Red®, TAMRA, JOE, Rhodamine (R6G) or ROX, BODIPY® and other dyes (Alexa®, Marina Blue®, etc.). These labels can be linked internally to any dT-residue, dR-residue or to either terminus. The fluorescent molecule is linked to the oligonucleotide via a spacer (a C-6 spacer generally) to limit the steric hindrances. Automatic coupling Automatic coupling uses the appropriate phosphoramidite to which the dye is already coupled like Cy® dyes, FAM, Fluorescein, HEX, TET and Yakima Yellow®. These labels are coupled during the synthesis at the 5' terminus. The easiest probes to synthesize and to purify are Double-Dye Oligonucleotide probes, as they are not too long. Moreover, the probes where you can use phosphoramidites (automatic coupling method) are usually the easiest ones to synthesize and purify, as there is only one set-up for the synthesis and one purification step. They also give the highest yields. The probes, which require post-labelling procedures (manual coupling method) are more laborious to synthesize and will generally yield lower amounts of product. Purification level Regarding the quality, it is recommended to work with purified primers for qPCR assays (Cartridge Reverse Phase purified or HPLC purified primers are ideal). The first aim of any purification method is to remove the by-products, resulting from the synthesis step (mainly salts). The next goal is to enrich the product in full-length oligonucleotides. Consequently, purified primers are more adapted to the sensitive qPCR tool than simply desalted primers. It is particularly true when working with non-specific detection systems like SYBR® Green. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 27 GUIDELINES FOR PRIMERS AND PROBES DESIGN General rules Well-designed primers and probes are a prerequisite for successful qRT-PCR. By using well-designed primers and probes, PCR efficiencies of 100 % can be obtained. If the following primer and probe design guidelines are taken into account you will achieve high PCR efficiencies, specific PCR products, non co-amplification of gDNA and therefore the most sensitive results. We do recommend in general using a design software (for example Oligo® Primer Analysis Software) to check for all following criteria. Most thermocycler softwares now offer tools to help you designing primers with the best characteristics. Some of the best softwares are Beacon Designer, Primer Express, and DNA Star... Some other tools are freely available on the web, for example: • http://medgen.ugent.be/rtphmerdb/ (human primer and probe database) • http://frontend.bioinfo.rpi.edu/applications/mfold/ (for testing secondary structures) • http://www.ebi.ac.uk/~lenov/meltinghome.html (Tm calculators) • http://frodo.wi.mit.edu/cgi-bin/primer/primer3_www.cgi (Design Primer, also hybridization probes) • http://bibiserv. techfak. uni-bielefeld. de/genefisher2 (Design Primer including degenerate primers) • http://www.premierbiosoft. com/qpcr/index Primers or SNP databases are also freely available, for example OMIM (from the NCBI), SNPedia, Huge Navigator and RT Primer DB (Pattyn F. ef a/., 2006) EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Design guidelines for SYBR® Green I assays As SYBR® Green I binds to any dsDNA it is important to avoid primer-dimers and non-specific products in SYBR® Green I assays. This can only be avoided by carefully selecting primers that only bind to the selected target. By selecting amplicons between 100 and 150 bp a high level of fluorescence can be obtained without compromising the PCR efficiency. One important characteristic of primers and probe is the Tm (melting temperature), the temperature at which 50 % of the oligonucleotide is hybridized. The simplified calculation method for the Tm is Tm=2(number A+T)+4(number G+C). Primers • Length : 18-30 bases • GC content: 30-80 % (ideally 40-60 %) • Tm : 55-60 °C, ATm difference between forward primer and reverse primer should be < 4 °C • Avoid mismatches between primers and target, especially towards the 3' end of the primer. Avoid runs of identical nucleotides, especially of 3 or more Gs or Cs at the 3' end. • Avoid 3' end T (allows mismatching) • Avoid complementarities within the primers to avoid hairpins • Avoid complementarities between the primers to avoid primer dimers, especially of 2 or more bases at the 3' ends of the primers • Check if primers are unique and specific (check with a BLAST search: www.ncbi.nlm.nih.gov/BLAST/) • Design intron spanning or intron flanking primers to prevent amplification of contaminating genomic DNA. For intron spanning primers the first half of the oligo must hybridize to the 3' end of one exon and to the 5' end of the other exon. In this way only cDNA will be amplified and not gDNA (fig. 11). Intron spanning primers For ware t Reverse Exon 1 Exon 2 Exon 3 gDNA No amplification Forward cDNA Amplification Figure 11. Intron spanning primers EUROGENTEC | www.eurogentec.com | info@eurogentec.com 29 For intron flanking primers, the forward primer must hybridize to one exon and the reverse primer to the other exon. Amplicons from cDNA, without introns, will be smaller than the amplicons from gDNA, which will contain the intron. The bigger amplicon will be amplified less efficiently. The difference in size of these amplicons can be determined via meltcurve analysis. If genes of only one exon are studied contamination with gDNA can only be avoided by DNase treatment of the RNA with RNase free DNase (Vandesompele, 2002). A recent study indicates that primers with a 5' flap improve Real-Time PCR results (Afonina, 2007). A unique validated 12-bases long AT-rich sequence added to the 5' extremity of both primers might increase the level of fluorescence and improve the sensitivity of the assay. The addition of this sequence is claimed to be especially useful for short primers (when the design is constrained) or on damaged DNA (for example sodium bisulfite treated DNA). The positive effect has been observed both for SYBR® Green I and Double-Dye probe assays. Amplicon • Length : 80-150 bp Shorter amplicons will give higher PCR efficiencies Longer amplicons will give a higher ARn as more SYBR® green I is incorporated • GC content 30-80 % (ideally 40-60 %) • Avoid secondary structures in the amplicon (check with Mfold: http://frontend. bioinfo.rpi.edu/applications/mfold/). EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Design guidelines for Double-Dye probe assays In probe assays, primer dimers and non-specific products will not be detected, however, they will influence the PCR dynamics and efficiency. Therefore, in probe assays they should be avoided as much as possible. For probe assays the amplicons should be kept as short as possible, with the 5' end of the probe as close as possible to the 3' end of the forward primer in case the probe is on the same strand, and as close as possible to the 3' end of the reverse primer in case the probe is on the opposite strand. In this way the 5' nuclease reaction will be optimal. Experience has showed that it is easier to first design the probe, and then the primers, rather than the other way around. Primers and probes Primers Design rules for a probe assay are the same than for the SYBR® Green assay, see above. Probes • Length 18-30 bases, optimal length: 20 Lengths over 30 bases are possible, but it is recommended to position the quencher not at the 3' end, but internally 18-25 bases from the 5' end (normally coupled to a T) • GC content: 30-80 % • Tm of the probe must be 8-10 °C higher than the Tm of the primers (8 °C for genotyping, 10 °C for expression profiling) • Select the strand that gives the probe more Cs than Gs • Place the probe as close as possible to the primers without overlapping them • Avoid mismatch between probe and target • Avoid complementarities within the probe • Avoid runs of identical nucleotides, especially of 4 or more Gs • Avoid 5' end G (as this can quenche several fluorophores, including FAM) For multiplex assays / genotyping • Position the polymorphism in the center of the probe • Adjust the probe length so that both probes have the same Tm Amplicon • Length 80-120 bp optimal Shorter amplicons will give higher PCR efficiencies and more efficient 5' nuclease reactions • GC content: 30-80 % (ideally 40-60 %) Avoid secondary structures in the amplicon (check with Mfold: http://frontend.bioinfo.rpi.edu/applications/mfold). EUROGENTEC | www.eurogentec.com | info@eurogentec.com 31 Quenching principle Fluorophore and quencher: definitions Many commonly employed techniques for the detection of nucleic acid sequences in a homogenous manner use fluorescence as the signaling technology. Typically a single-stranded probe is labelled with a fluorophore and a quencher molecule. Changes in quenching of the fluorophore, caused by hybridization of the probe to its target nucleic acid, lead to signal generation. Thus, Real-Time PCR relies not only on the choice of one technology, but also in the choice of the right fluorophore-quencher pairs, whether the assay would be singleplex or multiplex. The choice of a fluorophore and its combination with a quencher, will give different results in terms of sensitivity of the assay. • A fluorophore is a molecule that emits light of a certain wavelength after having first absorbed light of a specific but shorter wavelength. The emission wavelength is always higher than the absorption wavelength. • A quencher is a molecule that accepts energy from a fluorophore in the form of light and dissipates this energy either in the form of light or heat. Different ways of quenching Quenchers are molecules that can accept energy from a fluorophore and dissipate it by two mechanisms, called proximal, and FRET quenching. A fluorophore absorbs light energy and is promoted to an excited state. In the absence of a quencher the fluorophore falls back to the ground state and the excess of energy is released as fluorescence. J- F Figure 12. Fluorescence principle Proximal quenching When the fluorophore is in close proximity of a quencher molecule, the energy is transferred from the fluorophore to the quencher, which then dissipates the energy as heat (no fluorescence is observed). It is also known as collisional quenching. This type of quenching is used in Molecular Beacons and Scorpions® primers. ř- G* Abi t= LÜ£Si_^> - Q Figure 13. Proximal quenching principle EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide FRET quenching The fluorophore transfers its energy to the quencher (which may be another fluorophore); the energy is released from the quencher as fluorescence of a higher wavelength. The efficiency of this process is dependent on the distance between fluorophore and quencher, more precisely on the Förster distance 1/r6 (where "r„ is the fluorophore - quencher distance). This type of quenching is used in Double-Dyes probes and LC Hybridization probes. Dark quenchers dissipate the energy as heat, resulting in an absence of fluorescence emitted by the quencher. This type of quenchers allows a better signal-to-noise ratio in the assay. Figure 14. FRET quenching principle Optimal fluorophore quencher combination The quencher has to fit the fluorophore One fluorophore can be combined with multiple different quenchers, but the absorption spectrum of the quencher needs to have good overlap with the emission spectrum of the fluorophore in order to achieve optimal quenching. • For example, Deep Dark Quencher II absorbs over a large range of the visible spectrum and therefore efficiently quenches most of the commonly used fluorophores, especially those emitting at higher wavelengths. • Deep Dark Quencher I and Eclipse® Dark Quencher, quench the lower wavelength dyes such as FAM but they are not good at quenching those that emit at high wavelengths such as Cy® dyes. • The Black Hole Quencher family covers a large range of wavelengths (over the entire visible spectrum and into the near-IR) and is consequently one of the best options for multiplex application. Quenchers have a quenching capacity throughout their absorption spectrum, but the performance is best close to the absorption maximum. Quencher Absorption (nm) Emission (nm) TAMRA 565 580 BHQ-1™ 534 none BHQ-2™ 580 none BHQ-3™ 670 none Deep Dark Quencher 1 430 none Deep Dark Quencher 1 630 none Dabcyl Table 3: Absorbance and emission wavelengths of common Quenchers (BHQ: Black Hole Quencher) EUROGENTEC | www.eurogentec.com | info@eurogentec.com 33 600 700 Wavelength [nml Emission maxima of common dyes: 1. FAM 2. Yakima Yellow® - VIC® 3. TAMRA 4. CY®3 5. ROX 6. CY®5 Figure 15. UV-vis absorbance spectra of Deep Dark Quencher (pink), DabCYL (grey), Eclipse1' Dark Quencher (purple) and Deep Dark Quencher II (orange) I 3 6 10 500 600 700 Wavelength (nm| Emission maxima of common dyes 1. FAM 2. TET 3. Yakima Yellow® h. HEX 5. Cy»3 6. TAMRA 7. Cy»3.5 8. ROX 9. Cy»5 10. Cy»5.5 BHQ-0" ■ BHQ-1™ ■ BHQ-2™ ■ BHQ-3™ Figure 16. UV absorbance spectra of the Black Hole Quencher" family EOOiI'p' tn » io lo 3d | i 40 50 Figure 19. Threshold definition and Ct value calculation The baseline is the average background. It is calculated according to the noise level in the early cycles, when there is no detectable increase in fluorescence, due to PCR products. When the baseline is set up manually, it is important to ensure that only cycles where there is no signal increase are selected ! The threshold is the level of fluorescence above the baseline, at which the signal can be considered not to be background. The threshold can be calculated automatically or can be set manually. The automatic calculation of the threshold corresponds to the average baseline + "X" standard deviation of this baseline. The Ct value (Cp for Roche's thermocyclers) is defined as the cycle in which there is a significant increase in reporter signal, above the threshold, i.e. the cycle in which the growth curve crosses the threshold. It is consequently related to the initial amount of DNA and shows also the sensitivity of the assay. The Ct value is consequently in inverse proportion to the expression level of the gene. If the Ct value is low, it means the fluorescence crosses the threshold early, meaning that the amount of target in the sample is high. The Ct is therefore dependent on the threshold level, and it is then important to compare the threshold value from one run to another, if no normalization method is used. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 39 The ROX is a fluorescent dye that is used as a passive reference for some thermocyclers (Eppendorf, ABI Prism®, Stratagene...). This dye is usually spiked into the MasterMix and the reporter fluorescence is normalized to the ROX signal on ABI and Stratagene Mx machines. The Rn (normalized reporter signal) is calculated by dividing the reporter signal by the ROX signal. This signal is then corrected for pipetting inaccuracies, variation in sample volume, bubbles in sample, plastics inconsistency... When correction for background variations is required, the ARn is used. The ARn is calculated by subtracting the normalized baseline from the normalized reporter signal. ARn = Rn-Bn where: Rn= (reporter signal / ROX passive reference signal) Bn= (baseline signal / ROX passive reference) Only a minor variation in the ROX passive reference will cause a change in the Rn, Bn, ARn, threshold and Ct value. Therefore we recommend using MasterMixes that already contain a ROX passive reference to avoid such variations, which might be interpreted as real differences, but in reality are just experimental artifacts. Normalization and quantification methods When analyzing and comparing results of Real-Time qPCR assays many researchers are confronted with several uncontrolled variables, which can lead to misinterpretation of the results. Those uncontrolled variables can be the amount of starting material, enzymatic efficiencies, and differences between tissues, individuals or experimental conditions. In order to make a good comparison, normalization can be used as a correction method, for these variables. There are several ways to normalize, which all have some advantages and disadvantages. The most commonly known and used ways of normalization are : normalization to the original number of cells, normalization to the total RNAmass, normalization to one or more housekeeping genes, and normalization to an internal or external calibrator. Normalization to number of cells can actually only be done for cell culture and blood samples. In solid tissues and tumors, the amount of cells cannot be counted, only estimated and therefore leads to inaccuracies (Bustin, 2000). Normalizing to the RNA mass quantity will lead to inaccuracies as the total RNA mass (rRNA, tRNA and mRNA) might contain varying imbalances between rRNA and mRNA (Solinas ef a/., 2001). The determination of the RNA amount by photospectrometry is another factor causing inaccuracy, as the A260 quantification is strongly influenced by the impurity of the sample, caused by contaminating DNA, free nucleotides and proteins (Bustin, 2000 - Bustin, 2002). As very little is known about total RNA content per cell (in different tissues), a variation between individuals and between normal or highly proliferating tissues, like tumors will cause unwanted inaccuracies (Bustin, 2000 - Bustin, 2002). EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Normalization to one housekeeping gene excludes some of the above-mentioned drawbacks, but is also not the perfect method. The advantage is that the variation due to different amounts of RNA can be excluded. However, one must assure oneself, that the housekeeping gene is expressed constantly at the same level throughout the experiment and between samples. It is better then to use several housekeeping genes. A housekeeping gene used for normalization can also be used as a control (cf. p. 45). The two majors methods of normalization are the absolute quantification and the relative quantification (Sellars MJ et a/., 2007). Figure 20 summarizes the quantification methods that will be developed further. In absolute quantification, the exact number of copies of the gene of interest is calculated. In relative quantification, the expression of the gene of interest in a sample is expressed relatively to another gene, another sample, used as a reference. Absolute quantification Relative quantification External calibration curve SYBR Green assay, with or without ROX normalization External calibration curve Probes assays, with or without ROX normalization External calibration curve without any reference gene Normalization with one or multiple reference genes Standard curve with: • RT-PCR product • synthetic RNA • synthetic RNA • synthetic DNA • plasmid • previous PCR product • pooled cDNA • gDNA Without Real Time PCR efficiency correction AACt method With PCR efficiency correction see formula in thetext Figure 20: Summary of quantification methods in Real-Time qPCR *^ EUROGENTEC | www.eurogentec.com | info@eurogentec.com 41 Absolute quantification Absolute quantification requires a standard curve of known copy numbers {cf. p. 46 for the construction and analysis of the standard curve). It can be constructed using several standards (Figure 21). Plasmid, linear PCR product Pooled cDNA Genomic DNA Commercial RNA InVitro Trans RNA None Plasmid, not linear Other Bacteria Plasma Plasmid + Stds Figure 21: Most frequently used quantification standards. From Nucleic Acid Research Group, (NARG) survey 2007, http://www.abrf.org/NARG/ The amplicon being studied can be cloned, or a synthetic oligonucleotide (RNA or DNA) can be used. For gene expression studies, it is recommended to use an RNA standard, so the quantification method takes into consideration any possible RT efficiency variation. The standard must be amplified using the same primers as the gene of interest and must amplify with the same efficiency. The standards must also be quantified accurately. This can be carried out by reading the absorbance at A260, although this does not distinguish between DNA and RNA, or by using a fluorescent ribonucleic acid stain such as RiboGreen. The problem when using in vitro transcribed RNA as standard is that the construction of cDNA plasmid has to be done and has to be reverse transcribed. Moreover, it is not very stable for long-term storage. The advantages of using cDNA plasmid standards is that cDNA plasmids are easy to construct, they can be prepared in large amount and can be stored for a very long time without any difficulties. Relative quantification Relative quantification is the most widely used technique. Gene expression levels are calculated by the ratio between the amount of target gene and an endogenous reference gene, which is present in all samples. The reference gene has to be chosen so that its expression does not change under the experimental conditions or between different tissues (Cook NL ef a/., 2008). There are simple and more complex methods for relative quantification, depending on the PCR efficiency, and the number of reference genes used. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Delta delta Ct (AACt) method This method is the simplest one, as it is a direct comparison of Ct values between the target gene and the reference gene. However, PCR efficiencies of both the target and of the reference gene should be close to 100 % and not differ by more than 10%. Only the initial experiment requires a standard curve to compare the PCR efficiency of the target and control gene (Schmittgen TD, ef a/., 2008). Relative quantification involves the choice of a calibrator sample. The calibrator sample can be the untreated sample, the time=0 sample, or any sample you want to compare your unknown to. Firstly, the ACt between the target gene and the reference gene is calculated for each sample (for the unknown samples and also for the calibrator sample). — target reference gene Then the difference between the ACt of the unknown and the ACt of the calibrator is calculated, giving the AACt value: — (^^target ^^reference^calibrator (^^target ^^reference^sample The normalized target amount in the sample is then equal to 2MCt and this value can be used to compare expression levels in samples. Different PCR efficiencies If there is more than 10 % difference in PCR efficiencies, between the reference gene and the target gene, then it is inaccurate to use the AACt method. The value used is then: Ratio = (E ) ACt target (calibrator - sample) / /p \ ACt reference (calibrator - sample) \jwhprp ■ \ target' \ referenced ^target= PGR efficiency of the target ACt target (calibrator-sample) = (Ct,arge.) in the Calibrator - (Ct^ in the Sample. When several reference genes are used As mentioned in the Normalization chapter, it is better to use several control genes, because the expression of one reference gene might change slightly. It is then necessary to use an index reference gene (REF), a method described by M. Pfaffl and J. Vandesompele, 2004. The normalization by geometrical averaging of multiple internal control genes (Vandesompele ef a/., 2002) is a robust and innovative approach for accurate normalization. It relies on the use of several housekeeping genes to even out variations in the expression of these genes. Only the most stable expressed housekeeping genes are taken into the calculation. The calculation can be performed using the freely available software geNorm (http://medgen.ugent.be/~jvdesomp/genorm/). The major drawback of this method is the need for many primer pairs and the complicated way to process the data. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 43 EVALUATION OF YOUR qPCR ASSAY Several parameters can help you validating your assay. As for any experiment, before using a qPCR test routinely, it is important to optimize it, and validate its reproducibility. Controls The optimal way of processing is to include several negative and positive controls. The main ones are shown on Figure 22. ,i Technical repli Q. Minus RT or RNA No Template Control (NTC) to check for c logical replicate (ie., different samples, same treatment) Standard curve :DNA sample following RTreactior checkfor genomic DNA contamination External (exogeneousl positive control RNA sample prior to RT reactior External (exogeneousl negative control Positive Control (IPC) to check for PCR inhibition None / Not applicable Frequency (1 =Least, 5=Most) Figure 22: Frequency of replicates controls. From Nucleic Acid Research Group, NARG survey 2007, http://www.abrf.org/NARG/ Negative Controls • NTC: the No Template Control contains water and reaction mix, instead of template and reaction mix. Of course you should not observe any amplification in the NTC. However, it is sometimes difficult to achieve. In SYBR® Green assays, if you have many constraints on the design of the primers, you might observe a late amplification due to primer dimers. In any case, the amplification in the non-template control should go up late, and be at least 8 Ct after your specific signal, thus avoiding false positive interpretation. If it is not possible to achieve such a result, it is better to switch to a probe assay that is more specific. A minimum of 2 NTC's should be included. • NAC: the No Amplification Control includes all the components except the Polymerase. It is not possible to do one if you use a MasterMix that contains everything already. However, you can add only probe, primers, template and water, to check the integrity of the probe. If you observe an increase of fluorescence in this case, it is most likely that the probe is degraded. The NAC is less important than the other negative controls, as the probe degradation might also be detected with a high background level. • No RT: the No Reverse Transcriptase control is a sample without the addition of the Reverse transcriptase prior to the PCR step. If the No RT control shows an amplification (and not the NTC control), you can conclude that contaminating genomic DNA is present. The best way to proceed then is to design intron spanning primers or intron flanking primers. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl Positive controls Their main goals are to detect the quality of the reagents used, the presence of inhibitors in your sample, or damaged sample. Positive controls can also be used for normalization. Endogenous Positive Control An endogenous control is a second target for which the expression level is measured in the same sample than the Gene of Interest (GOI=target). In can be run in multiplex or in a different well. Endogenous positive control gives indication on the buffer or polymerase action. An endogenous control gene can be used to standardize the amount of sample RNA or DNA added to the reaction. It should be a stable expressed gene through the experiment, that is used to normalize the results of a variable target gene, and to correct for sample-to-sample variations. Exogenous Positive Control It is a different sample in which, the gene of interest is known to be expressed. It can be a previous PCR product, a plasmid containing the gene of interest, in vitro RNA transcripts... Thanks to this control, it is possible to check the quality of the primers, probe, and reagents but won't give any indication concerning the sample (degradation, inhibitors...). Spiking Control or Internal Positive Control (IPC): it is a DNA or RNA that is spiked into the sample and amplified using specific primers. Any variation of this spiking control expression would show that inhibitors are present in the sample (Scipioni A. ef a/., 2008). qPCR controls © NEGATIVE AIM • No Template Control Detection of primers dimers and contamination • No Amplification Control Detection of probe's degradation • No RT Control Detection of genomic DNA contamination © POSITIVE AIM • Endogenous Control (same sample, different target) Check quality of reagents. Also used for normalization. • Exogenous Control (same target, different sample) Check quality of reagents. Spiking Control (additional DNAspiked intothesample, different target) Detect inhibitors presence Reject false negative in diagnostic assays Figure 23. Summary of the main existing controls in qPCR and their interest. *^ EUROGENTEC | www.eurogentec.com | info@eurogentec.com 45 Commonly used control kits A good control should have a constant level of expression between individuals, among different tissues of an organism, at all stages of development, and should not be affected by the experiment treatment. The control gene should also be expressed at similar level as the gene of interest and the range of linear amplification should be known (Bustin, 2000). If a control gene is up and down regulated by the experimental intervention, this will lead to an incorrect normalization and thus to misinterpretation of the results. Therefore, proper validation of presumed stability of expression of the control genes, should be done before studying the target gene. The table below describes the expression level of commonly used control genes. Symbol Accession number Name Pseudogene Relative expression level M10098 18S rRNAO Very high M11167 28S rRNAO Very high ACTB NMJM101 Beta actin + High GAPD NM_002046 Glyceraldehyde-3-phosphate dehydrogenase + High UBC M26880 Ubiquitin C High B2M NMJM3194 Beta-2-microglobulin High YWHAZ NMJM0190 Tyrosine 3-monooxygenase activation protein, Zeta polypeptide (Phospholipase A2) + Medium RPL13A NMJ12423 Ribosomal protein L13a + Medium SDHA NMJM4168 Succinate dehydrogenae complex, subunitA + Medium HPRT1 NM_0000194 Hypoxanthine photshoribosyl-transferase I + Medium TBP NMJM3194 TAT box binding protein Low HMBS NMJM0190 Hydroxy m ethyl-bilane synthase Low Table 5. Relative expression level and presence (+) or absence (-) of pseudogenes in the genome concerning the most commonly used control genes Singleplex or multiplex assay for the controls? The endogenous control can be quantified in a singleplex reaction. The gene of interest and control are, in this case, amplified in separate reactions, using the same cDNA sample. It can also be used in a multiplex reaction, which prevents pipetting errors between the control reaction and gene of interest reaction, which can occur when using singleplex reactions. In this case, normalizing in the same well, will account for the variability in individual PCR assays, like inhibitors. Moreover, multiplex assays have the advantage of being cost effective in terms of reagents required. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Role of the standard curve and evaluation of its parameters Preparing a standard curve for each gene, which needs to be analysed, can provide a good idea of the performance of the qPCR. The standard curve should cover the complete range of expected expression. Using standard material (plasmid, PCR product etc.), the standard curve should include at least 5 points of dilution, each of them in duplicate (at least). The 10-fold or 2-fold dilution range should cover the largest range of expression levels. Plotting these points on a standard curve, will determine the linearity, the efficiency, the sensitivity and the reproducibility of your assay. Standard Curve Std. curve samples 21 20 Point 19 Ol _£ 0 Ü 17 16 -1 0 Log Concentration Figure 24: Example of a 2 times-serial diluted cDNA standard curve. Detection using SYBR® Green I on a LC480 thermocycler. PCR efficiency The slope of the standard curve gives the efficiency of the PCR reaction by the following equations: Exponential amplification = 1 o(-1/slope| Efficiency = 10(-1/slope| -1 If the slope of the standard curve is -3.32 then the PCR is 100 % efficient. With 100 % efficiency, a 2x dilution gives a ACt of 1 between each dilution (each cycle the amount of amplification is doubled). With a 100 % efficiency, a 10x dilution gives a ACt of 3.2 values between each dilution (every 3.2 cycles the amount of amplification is 10 fold higher). PCR efficiency between 90 %-110 % is acceptable (i.e. a slope between 3.1 and 3.58) EUROGENTEC | www.eurogentec.com | info@eurogentec.com 47 The following factors influence the PCR efficiency: • Length of the amplicon • GC content of the amplicon • Secondary structures in primers, and / or probe, and / or amplicon • Concentration of reagents R square (R2) The R2 is a parameter, which indicates how well the data points lie on line. It shows consequently the linearity of the PCR assay. If the R2 < 0.95 there is an indication that either the reactions have not been pipetted accurately, or that there is no linear relation between the Ct and the 10 log of the DNA concentration. The latter can be caused by inhibitory factors that are diluted out. A R2 higher than 0.985 is acceptable. Sensitivity You can compare the sensitivity of your assay by comparing the Ct values. The lower your Ct value is, the higher your assay sensitivity. However, be careful when you compare an assay in terms of sensitivity. You must compare it with the same sample, under the same running conditions, using the same threshold level. A dispersion of the duplicates/triplicates at the lower concentrated sample on the standard curve, can also indicate a lack of sensitivity. Reproducibility The reproducibility is indicated by the replicates. The standard deviation between the replicates can be calculated. It's ideally better to avoid replicates that have more than 0.5 Ct of difference Specificity of a SYBR® Green I assay For SYBR® Green I assay, an additional point has to be taken into consideration for the evaluation of the assay, this is the specificity. At the end of the run, it's better to systematically run a meltcurve. The temperature is slowly increased from 60 °C to 95 °C whilst continuously monitoring the fluorescence. At a certain temperature, the whole amplified product will fully dissociate, resulting in a drop of fluorescence as the SYBR® Green dissociates from the dsDNA. As the temperature of dissociation is dependant on the length and composition of the amplicon, it is consequently possible to check how many products of amplification are present in the well. A nice meltcurve should show a unique dissociation peak (figure 25). If primer dimers are amplified, a small product is usually observed around 70 °C (figure 26). The primer dimer peak can sometimes even be higher than the peak of the specific product. The primer dimer peak will also be observed even in the NTC control, reinforcing the conclusion that the amplification is non-specific. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide IhHkahilu djhh-1 -« inn — Hirivn -m nn-1 ar hn._ WIH..KI Figure 25. Clean meltcurve (without any primers dimers) 3 2 0 — 8 - 2 2 I— 2 ^ 2 4 -2 -0 - L ce-d(F1 8 -4 - /A / \ C i a u 1 2 - / \ 8 - / \ O D 0 LL" 0 0 4 -2 - \ -0 2 - 67 1 1 1 1 3 68.0 70.0 72.0 74.0 i i i i 76.0 78.0 80.0 82.0 84 Temperature (°C) I 0 86.0 8 1 1 1 0 90.0 92.0 93.0 Figure 26. Meltcurve with primers dimers (corresponding to the first peak around 74 °C) EUROGENTEC | www.eurogentec.com | info@eurogentec.com 49 OPTIMIZATION TIPS In general If the PCR efficiency is drastically lower than 100 % we would recommend the following: • Check the primer design and optimize the primer concentration Trying a new design is sometimes the quickest and cheapest option. To optimize an assay, it is also important to determine the best primer and probe concentration. We recommend testing a matrix of primer and probe concentrations. For SYBR® Green assay: test primers at 50 nM, 100 nM and 300 nM final concentration, each one independently of the other. For probe assay, test primers at 100 nM, 300 nM and 900 nM and test probes at 100 nM and 250 nM. The best combination of forward primer, reverse primer and probe concentrations can then be selected. • Optimize the temperature reaction and times Try to change annealing temperature. Start at 60 °C, and then try 58 °C, 56 °C, 54 °C. Try also to increase it up to 64 °C. This annealing temperature of course depends of the Tm of your primers. • Go for a 3-step instead of a 2-step protocol In the following cases we recommend performing a 3-step protocol to obtain better results: if results show late Ct values, if the amplicon is quite long, if the results give a less steep growth curve, or if it is not possible to re-do the design. The protocol will then be as follows: 40 cycles Denaturation 95 °C for 15 seconds Annealing 60 °C for 30 seconds Extension 72 °C for 30 seconds Extension time can be increased with 10-second steps. It is also possible to add a data collection step after the extension step. To do so, ensure that the detection temperature is at least 3 °C lower than the Tm of the amplicon. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide For probe multiplexing assay/qualitative qPCR General way of processing for a multiplex assay: • Check carefully the possible complementarity between probes and primers for the different targets. • Perform (with each target) a singleplex qPCR, with a standard curve to calculate the PCR efficiency. • Optimize individually each singleplex assays to reach a PCR efficiency as close to 100 %. Multiplex and calculate again the PCR efficiency and the sensitivity of each target. You should not observe a big switch when comparing singleplex and multiplex results for the same target. If there is a loss of sensitivity/efficiency between the singleplex and the multiplex experiment, then try the following things, in this order: • Limit the probe/primer concentration of the most abundant target. • Optimize the annealing temperatures by trying steps of 2 °C higher and lower than the current annealing temperature. • Increase the primer/probe concentration of the less abundant target. It is actually important to do it in this order, as the main goal is to keep the total primers/ probe concentration the lowest possible. A high concentration could partially inhibit the Taq polymerase activity. Such problems can arise when amplification of both genes compete for the PCR reagents. A highly abundant gene can out-compete a less abundant template leading to a bias in the results. Limiting the primer concentration for the most abundant template can avoid this competition. However, this takes a considerable amount of development. Furthermore, even when working with limited primer concentrations, the detection of rarer target genes may not be possible or may be detectable at much reduced efficiency. Optimizing can also mean adding more units of Taq polymerase per reaction and therefore increasing the costs again. • Be more flexible with your design by using LNA® bases. By increasing the Tm of the probe, LNA® probes size can be reduced (cf. p. 15) and specificity increased. • Use a Dark Quencher: when multiplexing, the number of probes in the well increases, and this can lead to an increase of basal fluorescence. Moreover, quenchers such as TAMRA are also fluorophores, leading to high background fluorescence. That is why we recommend the use of Dark Quencher for multiplex assays. There are now several 'Dark quenchers' available. These absorb the energy emitted by the fluorophore, but release it as heat rather than fluorescence, thus leading to a better signal to noise ratio (cf. "Quenching principle"). Dark quenchers are completely non-fluorescent, and have an extremely low background. Using dark quenchers significantly improves signal to noise ratios (higher ARn, a lower background), and thus give a higher sensitivity in multiplex PCR. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 51 TROUBLESHOOTING GUIDE Contamination With genomic DNA The best proof of contamination with genomic DNA is a clean NTC, and a No RT control showing amplification. In this case, carry out a fresh extraction and RT step. If your sample is rare and precious, then use intron spanning primers (cf. p. 28). From a previous PCR run When using MasterMixes containing a blend of dUTPs and dNTPs, you can easily get rid of a contamination from a previous PCR run. Just use the UNG at the beginning of each run, this will destroy any PCR product containing dUTPs. Bad PCR efficiency due to primer dimers Primer dimers in SYBR® Green I assays are easily identifiable on the meltcurve, and the consequence would be a bad PCR efficiency. If you have the choice of the region in which you can design your primers, then the best way to proceed is to do another design. The time wasted for the achieved doubtful result during optimization of an assay with badly designed primers costs much more than a new pair of primers. If you are limited in the choice of the region for the design, then try to decrease the concentration of primers. You can also try different MgCI2 concentrations. The best way to proceed however would be to switch to a probe assay which can be more specific. Inhibition Decreasing the amount of template can identify the presence of PCR inhibitors in the template. If the Ct values decrease when decreasing amounts of template are used (inverted result), then, it is usually characteristic of the presence of inhibitors. Using an external spiking control can also show the presence of inhibitors. How to get rid of inhibitors? There are now several commercial kits to purify RNA samples on columns. You can also try an ethanol precipitation. If your target is expressed in a sufficient amount, the best way of processing is to dilute the cDNA sample. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide Strong background A strong background can be due to the degradation of the probe. Check it on the NTC, and/or on the multicomponent view, to see if the fluorescence increases without target. A strong background can also be due to the quencher noise, as quencher such TAMRA is also a fluorophore. Use Dark Quencher, especially for multiplex assays. A high concentration of DNA can also lead to a strong background, if you do multiplex, try to decrease the concentrations of individual primers and probes. A strong background can also be explained by simple reasons such as fingerprints on the plate, dirt in the thermocycler block, particles in the tube etc. Genotyping troubleshooting Probe cross hybridization or spectral overlap means that, for example, a X probe cross-hybridizes to the Y amplicon. The following solutions can be used: • Reduce the concentration of the "contaminating" probe, start at 300 nM and decrease to 50 nM. • Optimize the annealing temperature. A probe that hybridizes despite the SNR might not anneal at the optimal temperature. Carry out the same test by decreasing and increasing the annealing temperature by 2 °C-steps. • Increase the concentration of the probe that gives the lowest signal. • Use LNA® to have a more specific X probe, for example by surrounding the SNP by three LNA® bases. It can also be observed that the VIC/Yakima Yellow probe signal overlaps the FAM one, or the contrary. The same solution mentioned above can be tried. You might also try to reverse the fluorophore keeping the same sequences, or to redesign the probes. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 53 FREQUENTLY ASKED QUESTIONS Is a probe assay more sensitive than a SYBR® Green assay? Both assays can be equally sensitive, however the probe assay is more specific. Consequently, for difficult to optimize PCR assays, it is recommended to use a probe assay. How can I get rid of a contamination from a previous PCR product? Use a kit containing dUTP/dNTP and UNG, which will destroy previous PCR products at the beginning of each PCR program. Why is my Negative Control, positive? • Simple contamination by pipetting over the NTC wells: place them as far as possible from the lowest diluted sample (highest concentrated sample). • The water is contaminated: use freshly purified water • There are primer dimers in the SYBR® Green assay, leading to a late amplification. This can be checked on the meltcurve. • The probe is degraded. How can I avoid gDNA being amplified in a reaction, in which I want to quantify the amount of mRNA? Perform a DNase treatment of the sample, or design intron spanning primers. How can I avoid the formation of primer dimers? Use a HotStart enzyme, optimize the design, and reduce the primer concentration. Can I use primers I designed for classical PCR for my qPCR experiment? Some design rules are similar, however one major change is the length of the amplicon, which should ideally not exceed 150 bp in qPCR and can be much longer in PCR. Why do I have several peaks on my meltcurve? A peak before the specific peak is usually primer dimers. Two distinct melting peaks can be due to co-amplification of genomic DNA and cDNA, or can be due to a non specific product. What amount of template should I use in each well? 50 ng of template per well is usually enough. The maximum recommended amount of cDNA is 500 ng in 50 ul, as a too high quantity of RNA/cDNA might inhibit the qPCR reaction. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl qPCR guide How can I set up the threshold for my qPCR assay? The threshold, as the baseline, is set up automatically on most machines, and calculated during the early cycles of the qPCR run. However, you can also set it up manually. What is an acceptable PCR efficiency for my assay? A PCR efficiency between 96 % and 104 % is ideal. However a PCR efficiency between 90 % and 110 % is acceptable, and the assay can usually be optimized to achieve this. What is an acceptable R2 value for my assay? A R2 higher than 0.985 is acceptable. Should I use a kit containing ROX or not? It depends on your machine. The ROX passive reference is mainly used on the ABI Prism® and Eppendorf machines to normalize the signal. Check our thermocycler-kit compatibility table to be sure you use the right kit for your machine type. What should I give Eurogentec to get optimization / troubleshooting advice on my results? Try to sum up the aim of your qPCR assay and the species on which you work. Then you should send us the plate set up, the thermal cycling conditions, the kit and machine you use, the amplification curve and associated Ct values, the standard curve, the meltcurve for SYBR® Green assay, the raw data and multicomponent view if possible on your machine, the sequence and Tm of your primers/probe, the concentration of primers/probe, and the normalization method. Please send all this informations to qPCR@eurogentec.com. How should I store kits, primers and probes? Probe MasterMixes are stable for 2 years at -20 °C, SYBR® Green MasterMixes are stable for 1 year at -20 °C It is also possible to store them at 4 °C for one month. Avoid doing more than 5 cycles of freeze-thawing. RT mixes are stable for one year at -20 °C or -80 °C Probes and primers in solution should also be stored at -20 °C in the dark, and are stable for months. In which buffer should probes be resuspended? Probes are very sensitive to hydrolysis, so resuspend them in TE 0.01 or just water. Any acidic solution will hydrolyze the probes and give a lower signal to noise ratio. In which case is a one-step qRT-PCR recommended? For high-throughput screening and optimized reactions, and to avoid any contamination, a One-Step experiment is the best way to proceed. EUROGENTEC | www.eurogentec.com | info@eurogentec.com 55 EUROGENTEC PRODUCTS Kits and consumables Our MasterMixes are developed to work with standard temperature profiles, to be completely adapted to any assay. However, it is recommended to generate a primer optimization matrix and a primer and probe ratio matrix, as this is a crucial step to obtain the lowest signal-to-noise ratio, the earliest Ct value and to save maximum reagents. If you don't find the reagent you need in the table below, please contact us at qPCR@eurogentec. com. EUROGENTEC | www.eurogentec.com | info@eurogentec.com ^jl m c o a m m O c x: c x: cc qPCR Kits for SYBR® Assays • Compatibility with Real-Time thermocycler ABI GeneAmp® SDS 5700 ABI Prism* SDS 7000 ABI Prism* 7300/7700 ABI Prism* 7900HT StepOne* Plus MasterCycler® ep realplex ABI Prism® 7500 iCycler iQ* MyiQ* iQ5 Mx4000* MX3000P* MX3005P* with ROX normalisation Mx4000* MX3000P* MX3005P* without ROX normalisation Rotor-Gene" 2000/3000/6000 MiniOpticon® DNA Engine Opticon* 1 & 2 Chromo 4* CFX96 Smartcycler* 1 & 2 Quantica* LC480 qPCR MasterMixes with ROX passive reference Reference #RXNs 25 pi Sample reference qPCR MasterMix for SYBR* Green I (5 x 1.5 ml) qPCR MasterMix Plus for SYBR* Green I dTTP, 7.5 ml qPCR MasterMix Plus for SYBR* Green I w/o UNG, 7.5 ml RT-SN2X-03T RT-SN2X-03+WOUN RT-SN2X-03WOU+ 600 600 600 RT-SN2X-005+ (1) (1) qPCR MasterMix Plus 7,5ml for SYBR* Green I RT-SN2X-03+ 600 RT-SN2X-005+ qPCR MasterMix Plus 15 ml for SYBR* Green I RT-SN2X-06+ 1200 RT-SN2X-005+ qPCR MasterMix Plus 50 ml for SYBR* Green I RT-SN2X-20+ 4000 RT-SN2X-005+ One step qRT-PCR MasterMix for SYBR* Green 1,7.5 ml RT-SNRT-032X 600 RT-SNRT-0052X Two step qRT-PCR MasterMix for SYBR* Green I RT-SN2X-03RT 600 (1) MESA GREEN qPCR MasterMix Plus for SYBR* Assay dTTP, 7.5 ml RT-SY2X-03+WOUN 600 RT-SY2X-005+WOUN MESA GREEN qPCR MasterMix Plus for SYBR* Assay, 7.5 ml RT-SY2X-03+WOU 600 RT-SY2X-005+WOU MESA GREEN qPCR MasterMix Plus for SYBR* Assay, 15 ml MESA GREEN qPCR MasterMix Plus for SYBR* Assay, 50 ml MESA BLUE qPCR MasterMix Plus for SYBR* Assay, 7.5 ml RT-SY2X-06+WOU RT-SY2X-20+WOU RT-SY2X-03+WOUB 1200 4000 600 RT-SY2X-005+WOU RT-SY2X-005+WOU RT-SY2X-005+WOUB MESA FAST qPCR MasterMix Plus for SYBR* Assay, 7.5 ml RT-SY2X-03+WOUF 600 RT-SY2X-005+WOUF One step MESA GREEN qRT-PCR MasterMix for SYBR* Assay, 7.5 ml RT-SYRT-032X 600 RT-SYRT-0052X (1) Use the RT-SN2X-005+ sample to get a valid preview of the MasterMixes and Core Kits results •dTTP kit will perform slightly better that this sample kit • For RT reagents, combine this sample to ourRT enzyme (RT-0125-ER) by simply adding the RTto the MasterMix sample at the recommended concentration. qPCR Kits for SYBR® Assays • Compatibility with Real-Time thermocycler qPCR Core kits with ROX passive reference Reference #RXNs 25 til m c o a m m O ABI GeneAmp® SDS 5700 ABI Prism* SDS 7000 ABI Prism* 7300/7700 ABI Prism* 7900HT StepOne* Plus MasterCycler® ep realplex ABI Prism* 7500 iCycler iQ* MyiQ* iQ5 Mx4000* MX3000P* MX3005P* with ROX normalisation Mx4000* MX3000P* MX3005P* without ROX normalisation Rotor-Gene" 2000/3000/6000 MiniOpticon® DNA Engine Opticon® 1 & 2 Chromo 4® CFX96 Smartcycler® 1 & 2 Quantica® LC480 qPCR Core kit for SYBR SYBR® Green I RT-SN10-05 1000 (1) qPCR Core kit for SYBR SYBR® Green I dTTP RT-SN10-05NU 1000 (1) Two step qRT-PCR Core kit for SYBR® Green I RT-SNRT-05 1000 (1) qPCR MasterMixes with Low ROX passive reference qPCR MasterMix Plus for SYBR® Green I Low ROX, 7.5 ml RT-SN2X-03+WOULR 600 / MESA GREEN qPCR MasterMix Plus for SYBR® Ass; iy Low ROX, 7.5 ml RT-SY2X-03+WOULR 600 RT-SY2X-005+WOULR MESA GREEN qPCR MasterMix Plus for SYBR® Ass; iyLow ROX, 15 ml RT-SY2X-06+WOULR 1200 RT-SY2X-005+WOULR MESA GREEN qPCR MasterMix Plus for SYBR® Ass: iy Low ROX, 50 ml RT-SY2X-20+WOULR 4000 RT-SY2X-005+WOULR MESA BLUE qPCR MasterMix Plus for SYBR® Assay ' Low ROX, 7,5 ml RT-SY2X-03+WOULRB 600 RT-SY2X-005+WOULRB MESA FAST qPCR MasterMix Plus for SYBR® Assay Low ROX, 7,5 ml RT-SY2X-03+WOULRF 600 RT-SY2X-005+WOULRF qPCR MasterMixes without ROX passive reference qPCR MasterMix Plus for SYBR® Green I No ROX, 7.5 ml RT-SN2X-03+NR 600 RT-SN2X-005+NR Two step qRT-PCR MasterMix for SYBR® Green I No Rox RT-SN2X-03NRRT 600 (2) MESA GREEN qPCR MasterMix Plus for SYBR® Ass; iy No ROX, 7.5 ml RT-SY2X-03+NRWOU 600 RT-SY2X-005+NRWOU MESA GREEN qPCR MasterMix Plus for SYBR® Ass: iy No ROX, 15 ml RT-SY2X-06+NRWOU 1200 RT-SY2X-005+NRWOU MESA GREEN qPCR MasterMix Plus for SYBR® Ass; iy No ROX, 50 ml RT-SY2X-20+NRWOU 4000 RT-SY2X-005+NRWOU (1) Use the RT-SN2X-005+ sample to get a valid preview of the MasterMixes and Core Kits results • dTTP kit will perform slightly better that this sample kit • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RTto the MasterMix sample at the recommended concentration. (2) Use the RT-SN2X-005+NR sample to get a valid preview of the MasterMixes and Core Kits results • dTTP kit will perform slightly better that this sample kit • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RT to the MasterMix sample at the recommended concentration. qPCR Kits for SYBR® Assays • Compatibility with Real-Time thermocycler ABI GeneAmp® SDS 5700 ABI Prism* SDS 7000 ABI Prism* 7300/7700 ABI Prism* 7900HT StepOne* Plus MasterCycler® ep realplex ABI Prism* 7500 iCycler iQ* My iQ* iQ5 Mx4000* MX3000P* MX3005P* with ROX normalisation Mx4000* MX3000P* MX3005P* without ROX normalisation Rotor-Gene" 2000/3000/6000 MiniOpticon® DNA Engine Opticon® 1 & 2 Chromo 4® CFX96 Smartcycler® 1 & 2 Quantica® LC480 qPCR MasterMixes without ROX passive reference Reference #RXNs 25 pi Sample reference MESA BLUE qPCR MasterMix Plus for SYBR* Assay No ROX, 7.5 ml RT-SY2X-03+NRWOUB 600 RT-SY2X-005+NRWOUB MESA FAST qPCR MasterMix Plus for SYBR* Assay No ROX, 7.5 ml One step MESA GREEN qRT-PCR MasterMix for SYBR Assay No ROX, 7.5 ml RT-SY2X-03+NRWOUF 600 RT-SY2X-005+NRWOUF RT-SYRT-032XNR 600 (a) (b) (a) RT-SYRT-0052XNR qPCR Core kits without ROX passive reference qPCR Core kit for SYBR Green 1 No ROX Two step qRT-PCR Core kit for SYBR* Green 1 No ROX RT-SN10-05NR RT-SNRT-05NR 1000 1000 (a) (a) (b) (b) (a) (a) (1) (1) qPCR MasterMixes with fluorescein qPCR MasterMix Plus for SYBR* Green 1 with fluorescein, 7.5 ml RT-SN2X-03+NRFL 600 / MESA Green qPCR MasterMix Plus for SYBR* Assay w/ fluorescein, 7.5 ml RT-SY2X-03+WOUFL 600 RT-SY2X-03+WOUFL MESA Green qPCR MasterMix Plus for SYBR* Assay w/fluorescein, 15 ml RT-SY2X-06+WOUFL 1200 RT-SY2X-03+WOUFL MESA Green qPCR MasterMix Plus for SYBR* Assay w/ fluorescein, 50 ml RT-SY2X-20+WOUFL 4000 RT-SY2X-03+WOUFL MESA BLUE qPCR MasterMix Plus for SYBR* Assay w/fluorescein, 7.5 ml RT-SY2X-03+WOUFLB 600 RT-SY2X-005+WOUFLB (1) Use the RT-SN2X-005+NR sample to get a valid preview of the MasterMixes and Core Kits results. dTTP kit will perform slightly better that this sample kit. For RT reagents, combine this sample to our RT enzyme by simply adding the RT to the mastermix at the recommended concentration. (a) In combination with ROX passive reference • RT-PARE-03 (b) In combination with fluorescein additive • RT-FLUO-ADD qPCR Kits for Probe Assays • Compatibility with Real-Time thermocycler qPCR MasterMixes with ROX passive reference Reference #RXNs 25 pi m c o a m m O qPCR MasterMix, 5x15 ml qPCR MasterMix Plus, 7.5 ml qPCR MasterMix Plus, 15 ml qPCR MasterMix Plus, 50 ml FAST qPCR MasterMix Plus, 7.5 ml FAST BLUE qPCR MasterMix Plus, 7.5 ml qPCR MasterMix Plus w/o UNG, 7.5 ml qPCR MasterMix Plus, 7.5ml QGS qPCR MasterMix Plus dTTP, 7.5 ml FAST qPCR MasterMix Plus dTTP, 7.5 ml FAST BLUE qPCR MasterMix Plus dTTP, 7.5 ml One step qRT-PCR MasterMix, 7.5 ml Two step qRT-PCR MasterMix qPCR Core kits with ROX passive reference qPCR Core kit qPCR Core kit dTTP RT-QP2X-03 RT-QP2X-03-075+ RT-QP2X-03-15+ RT-QP2X-03-50+ RT-QP2X-03+F RT-QP2X-03+FB RT-QP2X-03WOU+ RT-QP2X-03+QGS RT-QP2X-03+WOUN RT-QP2X-03+WOUNF RT-QP2X-03+WOUNFB RT-QPRT-032X RT-QP2X-03RT RT-QP73-05 RT-QP10-05NU 600 600 1200 4000 600 600 600 600 600 600 600 600 600 1000 1000 ABI GeneAmp® SDS 5700 ABI Prism® SDS 7000 ABI Prism® 7300/7700 ABI Prism® 7900HT StepOne® Plus MasterCycler® ep realplex ABI Prism® 7500 iCycler iQ® MyiQ® iQ5 Mx4000® MX3000P® MX3005P® with ROX normalisation Mx4000® MX3000P® MX3005P® without ROX normalisation Rotor-Gene" 2000/3000/6000 MiniOpticon® DNA Engine Opticon® 1 & 2 Chromo 4® CFX96 Smartcycler® 1 & 2 Quantica® LC480 RT-QP2X-005+ RT-QP2X-005+ RT-QP2X-005+ RT-QP2X-005+ RT-QP2X-005+F RT-QP2X-005+FB RT-QP2X-005+WOU RT-QP2X-005+QGS RT-QP2X-005+WOUN RT-QP2X-005+WOUNF RT-QP2X-005+WOUNFB RT-QPRT-0052X (1) (1) Use the RT-QP2X-005+ sample to get a valid preview of the MasterMixes and Core Kits results • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RT to the MasterMix sample at the recommended concentration. qPCR Kits for Probe Assays • Compatibility with Real-Time thermocycler ABI GeneAmp® SDS 5700 ABI Prism* SDS 7000 ABI Prism* 7300/7700 ABI Prism* 7900HT StepOne* Plus MasterCycler® ep realplex ABI Prism* 7500 iCycler iQ* MyiQ* iQ5 Mx4000* MX3000P* MX3005P* with ROX normalisation Mx4000* MX3000P* MX3005P* without ROX normalisation Rotor-Gene" 2000/3000/6000 MiniOpticon® DNA Engine Opticon® 1 & 2 Chromo 4® CFX96 Smartcycler® 1 & 2 Quantica® LC480 qPCR Core kits with ROX passive reference Reference #RXNs 25 pi Sample reference qPCR Core kit only dUTP RT-QP10-05OU 1000 Two step qRT-PCR Core kit RT-QPRT-05 1000 (i) qPCR MasterMixes with Low ROX passive reference qPCR MasterMix Plus Low ROX, 7.5 ml RT-QP2X-03+WOULR 600 RT-QP2X-005+WOULR FAST qPCR MasterMix Plus Low ROX, 7.5 ml RT-QP2X-03+WOULRF 600 RT-QP2X-005+WOULRF FAST BLUE qPCR MasterMix Plus Low ROX, 7.5 ml RT-QP2X-03+WOULRFB 600 RT-QP2X-005+WOULRFB One step qRT-PCR MasterMix Low ROX, 7.5 ml RT-QPRT-032XLR 600 (2) qPCR MasterMixes without ROX passive reference qPCR MasterMix Plus No ROX, 7.5 ml RT-QP2X-03NR 600 RT-QP2X-005+NR FAST qPCR MasterMix Plus No ROX, 7.5 ml RT-QP2X-03+NRF 600 RT-QP2X-005+NRF FAST BLUE qPCR MasterMix Plus No ROX, 7.5 ml RT-QP2X-03+NRFB 600 RT-QP2X-005+NRFB One step qRT-PCR MasterMix No ROX, 7.5 ml RT-QPRT-032XNR 600 RT-QPRT-0052XNR Two step qRT-PCR MasterMix No ROX RT-QP2X-03NRRT 600 (3) qPCR Core kits without ROX passive reference qPCR Core kit No ROX RT-QP73-05NR 1000 (a) (3) Two step qRT-PCR Core kit No ROX RT-QPRT-05NR 1000 (a) (3) (1) Use the RT-QP2X-005+ sample to get a valid preview of the MasterMixes and Core Kits results • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RT to the MasterMix sample at the recommended concentration. (2) Use the RT-QPRT-0052XNR sample and add ROX passive reference to the reaction buffer at the recommended concentration (see ROXTDS). (3) Use the RT-QP2X-005+NR sample to get a valid preview of the MasterMixes and Core Kits results • For RT reagents, combine this sample to our RT enzyme (RT-0125-ER) by simply adding the RTto the MasterMix sample at the recommended concentration, (a) In combination with ROX passive reference • RT-PARE-03 m c o a m m O qPCR miscellaneous Fluorescein additive RT-FLUO-ADD 1 ml UNG (Uracil-N-Glycosylase) RT-0610-03 300 U ROX passive reference RT-PARE-03 300 til UNG (Uracil-N-Glycosylase) RT-0610-15 1500 U Reverse Transcriptase Core kit RT-RTCK-03 600 RXNs (25 til) EuroScript/RNAse inhibitor mix RT-0125-ER 3750 U Reverse Transcriptase Core kit RT-RTCK-05 1000RXNs(25tll) qPCR Internal Positive Control Internal Positive Control Yakima Yellow ** - TAMRA RT-IPCY-T02 200 RXNs (50 til) Internal Positive Control Yakima Yellow** - BHQI RT-IPCY-B02 200 RXNs (50 til) * For all One-Step RTkits ** Equivalent to VIC qPCR validated internal controls Target gene 18S rRNA 28S rRNA ß-actin GAPDH Ubiquitin C ß-2 microglobulin Phospholipase A2 HMBS RPL13a SDHA HPRT1 TBP Reference RT-CKFT-18S RT-CKYD-18S RT-CKYD-28S RT-CKYD-ACTB RT-CKYD-GAPD RT-CKYD-UBC RT-CKYD-B2M RT-CKYD-YWHAZ RT-CKYD-HMBS RT-CKYD-RPL13A RT-CKYD-SDHA RT-CKYD-HPRT1 RT-CKYD-TBP Relative expression level Very high Very high High High High High Medium Medium Medium Medium Low Low cDNA / / / / / / / / / / / / gDNA / / / / / / Organism H, Ra, M, Rb H,M H H H H H H H H H H Amplicon length 121 bp 87 bp 91 bp 86 bp 76 bp 112 bp 93 bp 119 bp 92 bp 118 bp 100 bp 145 bp H : human • Ra : rat • M : mouse • Rb : rabbit CKFT: FAM-TAMRA probe CKYD : Yakima Yellow (VIC® equivalent) - Eclipse® Dark Quencher probe Each kit contains enough reagents for up to 250 - 50 pi reactions. Other dye-quencher combinations are available on request. m c x o o m m O : o CC CC 96-well plate compatibility with Real-Time thermocyclers ABI GeneAmp8 5700 ABI Prism® SDS 7000 ABI Prism® SDS 7300 ABI Prism® SDS 7500 ABI Prism* SDS 7500 FAST ABI Prism® SDS 7700 ABI Prism® SDS 7900HT ABI Prism® SDS 7900 FAST ABI Step One ABI Step One Plus o 5 O tn S o Q CO o o "D CO o o cn "D MasterCycIer ep realplex DNA Engine Opticon® 1 DNA Engine Opticon® 2 Chromo 4 MiniOpticon® Quantica® o oo o qPCR 96-well plate type Cover Sub-skirted, high profile, frosted or white type (ABI systems) RT-PL96-AB Flat caps (RT-FLAT-300) •/ •/ -z •/ RT-PL96-ABW Adhesive seals (RT-OPSL-XX**)