Aplikovaná chemie a biochemie Přednáška č. 5 Nukleové kyseliny Práce s nukleovými kyselinami • Izolace DNA • Izolace RNA • Gelová elektroforéza • Detekce mRNA – Northern blotting, RNAse protection assay, RT-PCR, expression microarrays Izolace plazmidů RNA extraction and isolation “Isolation of high quality RNA is one of the most challenging techniques in modern molecular biology” Nice versus nasty Chomczynski, P. and Sacchi, N. Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate- phenol-chloroform extraction. Anal. Biochem. 162, 156-159 (1987). Will require use of inhibitors to inactivate endogenous RNases e.g. guanidinium isothiocyanate (GITC) RNAzol/Trizol/Ultraspec (14M GITC and urea salts) Qiagen RNeasy – use of columns to avoid organic extractions. Issues related to RNA isolation  protection of RNA against degradation by RNases  removal of contaminating DNA • acid-phenol chloroform extraction • oligo d(T) methods (only eukaryotic mRNA) • DNases • avoid touching samples • RNase inhibitors such as Guanidinium Thiocyanate Best methods for removing DNA contamination  Oligo d(T) methods (for eukaryotic mRNA only) • exploit the poly(A) tail of eukaryotic mRNA • hybridize Oligo d(T) + a with poly(A) tail • separate bound mRNA DNA bound mRNA pellet Best methods for removing DNA contamination  DNase 1. degrade contaminating DNA with Dnase 2. disable / remove DNase Izolace RNA pomocí kitů Total RNA versus mRNA Depends on final usage mRNA will comprise 2-5% of total RNA mRNA usually requires a further purification step – polyA tail Degradation can be visualised with total RNA RNA extraction and isolation Nové technologie pro analýzu čistoty a koncentrace NK NanoDrop® ND-1000 UV-Vis Spectrophotometer Small samples: designed for 1 ul samples Dynamic range: measures 2-3700 ng/ul (dsDNA) on a single sample. Full Spectrum (220-750nm) 10 second measurement time No cuvettes or capillaries Nové technologie pro analýzu čistoty a koncentrace NK Agilent 2100 Bioanalyzer http://www.chem.agilent.com/scripts/ge neric.asp?lPage=1565&indcol=N&prodcol= Y Metody analýzy RNA  RNA-targeted Fluorescent In Situ Hybridization • hybridization with anti-sense RNA  Ribonuclease protection assay • selection of desired RNA • protection against RNase  Northern blot analysis • RNA extraction, isolation & size fractionation Random priming: Labeling principle The method of “random primed” DNA labeling developed by Feinberg and Vogelstein (1,2) is based on the hybridization of a mixture of all possible hexanucleotides to the DNA to be labeled. All sequence combinations are represented in the hexanucleotide primer mixture, which leads to binding of primer to the template DNA in a statistic manner.Thus an equal degree of labeling along the entire length of the template DNA is guaranteed. The complementary strand is synthesized from the 3´OH termini of the random hexanucleotide primer using Klenow enzyme, labeling grade. Modified deoxy- ribonucleoside triphosphates ([32P]-, [35S]-, [3H]-, [125I]-digoxigenin or biotin-labeled) present in the reaction are incorporated into the newly synthesized complementary DNA strand. T4 nucleotide kinase: Labeling principle The bacteriophage T4 polynucleotide kinase catalyzes two reactions: forward and exchange. In the forward reaction, the enzyme transfers the phosphate of [-32P]ATP to the 5'hydroxy group of a DNA molecule (oligonucleotides or nucleoside 3'-monophosphates) or RNA, previously dephosphorylated with alkaline phosphatase. In the exchange reaction, T4 polynucleotide kinase transfers the 5'-terminal phosphate group of the DNA molecule to ADP. Then, the enzyme transfers the  phosphate of [-32P]ATP to the 5'-hydroxy group of a DNA molecule. 5 –10g isolated RNA 260/280nm ~ 2.0 (c.f. DNA 1.8) Northern Blotting Denaturing agarose gel electrophoresis Formaldehyde 0.66M/2.2mM Glyoxal/Methyl Mercuric Hydroxide Heat in formamide to denature Blot/transfer the RNA onto a membrane using salt solution RNA transferred to nylon or nitrocellulose membrane/filter Probe (cDNA fragment/antisense RNA) can be radiolabelled or chemiluminescent. Random priming is the usual method for generating a labelled probe Hybridization buffer is important as is stringency of washing (Rapid Hyb ~ 1hr) Northern Blotting Northern Blotting Probe can be stripped off the membrane which can be reprobed or multi-probed. Use a housekeeping type to probe to analyse changes Glyceraldehyde 3’ phosphate dehydrogenase (GAPDH), -actin, 2-microglobulin RNase protection assay The ribonuclease protection assay (RPA) is a highly sensitive and specific method for the detection of mRNA species. The assay was made possible by the discovery and characterization of DNAdependant RNA polymerases from the bacteriophages SP6, T7 and T3, and the elucidation of their cognate promoter sequences. These polymerases are ideal for the synthesis of high-specific-activity RNA probes from DNA templates because these polymerases exhibit a high degree of fidelity for their promoters, polymerize RNA at a very high rate, efficiently transcribe long segments. The strategy for the development of multi-probe RPA systems is to generate a series of related gene templates, each of distinct length and each representing a sequence in a distinct mRNA species. The templates are assembled into biologically relevant sets to be used by investigators for the T7 polymerase-directed synthesis of a high-specific-activity, [32P]labeled, antisense RNA probe set. The probe set is hybridized in excess to target RNA in solution after which free probe and other single-stranded RNA are digested with RNases. The remaining "RNase-protected" probes are purified, resolved on denaturing polyacrylamide gel, and quantified by autoradiography or phosphorimaging. The quantity of each mRNA species in the original RNA sample can then be determined based on the intensity of the appropriately-sized, protected probe fragment. Two distinct advantages of the multi-probe RPA approach are its sensitivity and its capacity to simultaneously quantify several mRNA species, in a single sample of total RNA. This allows comparative analysis of different mRNA species within samples and, by incorporating probes for housekeeping gene transcripts, the levels of individual mRNA species can be compared between samples. Advantages & disadvantages of RT-PCR  RT-PCR is very sensitive because of the ability to amplify product Northern blot 5 ~ 10mg RNasePA 100ng-1mg RT-PCR < 1ng (even single cells)  Able to look at multiple mRNA simultaneously by using multiple primers  May be too sensitive? Applications of RT-PCR  diagnosis of viruses  rRNA:rDNA ratio as an indicator of growth • universal activity • activity of specific organisms  RT in situ PCR • nifH gene  expression of specific mRNA as indicator of activity • degradation of xenobiotic compounds  expression of specific genes in specific tissues Reverse transcription (RT)-PCR Convert the RNA to cDNA using reverse transcriptase AAAAAAA 5’ 3’ Oligo dT TTTTTTT AAAAAAA 5’ 3’ Gene-specific primer AGCGA AAAAAAA 5’ 3’ Random primers (pdN6) NNNNNNNNNNNN Reverse transcription (RT)-PCR Reverse transcriptase - AMV or MMLV Typical RT reaction 1 g total RNA 1l 500ng primer (pdN6) 1l 0.1 mM DTT 1l 1mM dNTPs 2l 1U RNase inhibitor 1l Nuclease free H2O 13l 19l – heat 80C 4’, snap on ice + 1l 200U MMLV – superscript (Invitrogen) 25C - 10’ 42C – 50’ 95C - 2’ 1-20l into 50 l typical PCR Reverse transcription (RT)-PCR Note – the RT and PCR can be done in the same tube in a one-step reaction. Reverse transcription (RT)-PCR Semi-quantitative vs. quantitative Quantitative Real-Time PCR e.g. TaqMan Příklad: Kvatifikace CYP1A1 mRNA v buňkách ovlivněných PCB Induction of cytochrome p450 (CYP) 1A1 mRNA by PCBs in WB-F344 cells. (A) PCB 126 induced CYP1A1 mRNA expression in WB-F344 cells in a dose-dependent manner. Cells were treated with the indicated concentrations of PCB 126 or DMSO as a solvent control for 24 h. Total RNA was isolated and quantitative real-time RT-PCR was performed as described in the Materials and Methods. One representative experiment is shown out of three independent experiments. (B) Induction of CYP1A1 mRNA expression following the 24-h treatment of WB-F344 cells with PCBs (1 mM). (C) Expression of the reference gene porphobilinogen deaminase in samples analyzed in parts A and B. Current applications for DNA microarrays. Classical microarray experiments use isolated genomic DNA or mRNA from a whole organism or tissue. The DNA or mRNA is transformed and amplified into fluorescently labeled cDNA or cRNA, respectively, which is then hybridized to microarrays. These types of experiment have been used to identify changes in DNA copy number and mRNA expression patterns. Recent innovations in microarray approaches have used an additional purification step by protein immunoprecipitation to identify DNA (chromatin immunoprecipitation or ChIP) or mRNA-binding proteins. Protein bound to DNA or mRNA is first crosslinked and then immunoprecipitated by an antibody to a specific protein of interest. Crosslinks are then reversed, which releases the co-purified DNA or mRNA for amplification, labeling and hybridization to microarrays. These procedures have been successful in determining the targets of transcription factors, as well as genomic DNA-binding and mRNA-binding proteins. Microarray experiment. Plasmid clones are propagated in bacteria, and the cloned inserts are amplified by PCR and then purified. The purified PCR products are then robotically printed onto glass or nylon solid supports. Modifications of this approach include the use of oligonucleotides instead of PCR products or the in situ synthesis of oligonucleotides directly onto the glass support using photolithographic or other techniques. Separate nylon based arrays are hybridised with 33 P-radiolabeled cDNA prepared from the test and reference sample, whereas glass slide arrays are hybridised simultaneously with Cy5 and Cy3 fluorescently labeled test and reference samples, respectively. Following stringency washes, hybridisation to nylon arrays is detected by phosphorimaging. Hybridisation to glass slides is detected by excitation of the two fluors at the relevant wavelength and the fluorescent emission collected with a charge-coupled device. The test and reference images are overlaid using specialist software and can be displayed in a number of ways, including as a scatter plot of the ratio of test:reference gene expression. „„DiseaseDisease--specificspecific““ exprese genexprese genůů:: T.R. Golub et al., Molecular classification of cancer: class discovery and class prediction by gene expression monitoring, Science 286 (1999) 531– 537.