RNA in diagnostics • 1.) direct RNA diagnostics – screening of whole coding region of given gene • 2.) gene – expression analysis: – diferential diagnostics of some tumours – detection of circulation tumour cells in blood and bone marrow – monitoring of course of therapy and detection of residual disease – control of graft before autologous transplatation – differential display, PTT test, functional tests... RNA in diagnostics RNA Mammalian cell: • 10 - 30 pg total RNA • rRNA (28S,18S, 5S) 80-85% • tRNA, snRNA 15-20% • mRNA 1-5% 360 000 mRNA molecules/cell, 12 000 different transcripts typical length of 1 transcript cca 2kb • presence of ribonucleases (RNases) in cell • RNase – very stable – do not need cofactors – efficient in low concentrations – difficult inactivation – contamination with RNases : human skin dust particles (bacterias, fungi) • isolation and analysis of RNA : special approach and methods RNA Unstability •gene-expression analysis: analysed RNA must represent in vivo expression of sample •Complications - 1) reduction mRNA (downregulatoin of genes and enzymatic degradation of RNA), 2) expression induction of certain genes RNA stabilisation in sample: – immediately frost in liquid nitrogen and store in -80°C – stabilisation solutions: RNAlater, PAXgene Contamination with DNA PCR primers overlapping border intron/exon digestion with DNases Stabilisation and storage of RNA Direct RNA diagnostics RNA A AA A A A A T TT T TT T T TT T TT TcDNA PCR RT-PCR T TTT TT T primer oligo(dT) gene specific primer RNA Extraction NF1 gene: 350 kb, 60 exons, 11 - 13 kb mRNA - protein neurofibromin: 2818 aminoacids, probably tumour supresor RNA diagnostics of NF1 gene Neurofibromatosis type 1 von Recklinhausen disease Autosomal dominant Frequency 1:3000 Locus 17q 50% mutations de novo Predispositions to tumours of neural system Café-au- lait spots Neurofibromas Lisch nodule Complications in molecular diagnostics of NF1 -problematic clinical diagnosis: -up to 50 % cases de novo -high mutation speed -length of gene (350 kb, 60 exonů) -absence of hot spots – need to search in whole gene -unclear corelation between type of mutation and manifestation -different clinical manifestation even in patients with the same mutation Strategy of molecular – genetic testing of NF1 patients NF1 DNA / RNA of pacient Prenatal diagnostics intragene DNA polymorfismus IVS27AAAT2.1, IVS27AC28.4 IVS38GT53.0 - i 38 Linkage DNA analysis DNA/RNA methods DGGE, SSCP cDNA / SSCP Mutation analysis Direct sequencing cDNA - SSCP analysis RT cDNA PCR NF1 cDNA ( 60 exons) P1 P3 P5 P7 P9 P2 P4 P6 P8 P10 SSCP Sequencing analysis total RNA Sequencing of cDNA segment P7 of NF1 gene ( exons 28 -32/33) C >T cDNA of NF1 pacient, mt C5839T ( Arg > STOP) standard cDNA 5’ 3’ 5’ 3’ Advantages and disadvantages of RNA diagnostics - Easier and faster screening of multiexonic gene (10 segments of cDNA instead of 60 exons), mRNA is without introns - Capture of splice mutations in intrones - Capture of deletion of whole exonu on one allele - cheaper - More difficult taking blood for RNA isolation - Lower stability of RNA - Longer segments – problems with electroforetic separation and sequencing - Unclear effect of mutation on phenotype level Gene expression analysis Real-time PCR method  Real-Time PCR combines DNA amplification with real time amplified product detection in a single tube  Reliable  Precise  Fast  Universal  Variability of used probes  Possibility of detection of more mutation during analysis  Measurement of fluorescence Determine differences in mRNA expression  QUALITATIVE ANALYSIS snp – single ncleotide polymorphism detection  QUANTITATIVE ANALYSIS amplicon amount detection Detection methods  Two types of Real-Time PCR detection chemistries:  1. Specific Sequence Detection- Distinguishes between a specific sequence of interest and non-specific products. Can be used to detect different alleles (TaqMan)  2. Non-Specific Detection- Detects any dsDNA produced during the reaction (SYBR green) Tumour cells have different gene expression profile than healthy cells Change in amount and spectrum of expressed mRNA Relative quantification of expression relative to housekeeping gene Housekeeping gene: control gene which is constant in the samples Expression analysis in oncology Oncomarkers A tumor marker is a substance found in the blood, urine, or body tissues that can be elevated in cancer. There are many different tumor markers, each indicative of a particular disease process, and they are used in oncology to help detect the presence of cancer. From substances produced by normal cells they differ qualitatively – normal cells do not produce them, or quantitatively – they are produced by both types of cells • produced only in malignant cells • organ specific • in biological liquids in high concentrations • level correlates with size of tumour, stage of disease, prognosis and therapy effect • provides evidence of residual tumour tissue Ideal Oncomarker Oncomarkes according to chemical structure or biological function • oncofetal antigens • enzymes • hormones • intracellular oncomarkers • other non specified substances Onkomarkers: indication according to organ  Prostate-Specific Antigen: PSA is prostate-specific, not cancer-specific. A variety of conditions can raise PSA levels: prostatitis (prostate inflammation), benign prostatic hypertrophy (prostate enlargement), and prostate cancer. PSA levels can also be influenced by a number of other things.  Carcinoembryonic Antigen: Although CEA was first indentified in colon cancer, an abnormal CEA blood level is specific neither for colon cancer nor for malignancy in general. Elevated CEA levels are found in a variety of cancers other than colonic, including pancreatic, gastric, lung, and breast. It is also detected in benign conditions including cirrhosis, inflamatory bowel disease, chronic lung disease, and pancreatitis. The CEA was found to be elevated in up to 19 percent of smokers and in 3 percent of a healthy control population. Thus, the test for oncomarker cannot substitute for a pathological diagnosis. Detection of minimal residual disease Detection of presence of isolated tumour cells in blood, bone marrow and lymphatic system – possible precursors of metastases Imunohistochemistry – sensitivity 1 : 10 000 Flow cytometry – sensitivity 1 : 100 000 PCR – sensitivity 1: 1 000 000 Real- time PCR – sensitivity up 1 : 10 000 000 Human Identity Testing Applications: Forensic cases - matching suspect with evidence Paternity testing - identifying father Historical investigations Missing persons investigations Mass disasters - putting pieces back together Military DNA “dog tag” Convicted felon DNA databases Sources of Biological Evidence • Blood • Semen • Saliva • Urine • Hair • Teeth • Bone • Tissue Blood Stain: Only a very small amount of blood is needed to obtain a DNA profile Steps in DNA Analysis Collection Extraction Quantitation Genotyping Interpretation of Results Database Storage & Searching Specimen Storage DNA Database STR Typing DNA Extraction Multiplex PCR Amplification/RFLP Male: 13,14-15,16-12,13-10,13-15,16 Interpretation of Results Sample Collection & Storage Buccal swabBlood Stain DNA Quantitation Slot Blot 1 ng 0.3 ng 1 ng 1 ng 0.7 ng 0.5 ng 0.5 ng No DNA What Type of Genetic Variation? CTAGTCGT(GATA)(GATA)(GATA)GCGATCGT GCTAGTCGATGCTC(G/A)GCGTATGCTGTAGC •Length Variation short tandem repeats (STRs) •Sequence Variation single nucleotide polymorphisms (SNPs) insertions/deletions Example of three alleles: Allele 1: …AGA… Allele 2: …AGAAGAAGA… Allele 3: …AGAAGAAGAAGAAGA… Family Inheritance of STR Alleles (D13S317) Father Child #1 Child #2 Child #3 Mother PCR product size (bp) 11 14 11 12 14 8 14 12 128 Father Mother Paternity Testing Short Tandem Repeats (STRs) the repeat region is variable between samples while the flanking regions where PCR primers bind are constant AATG 7 repeats 8 repeats AATG Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another Primer positions define PCR product size Fluorescent dye label Fluorescent dye creates a labeled PCR product primer1 primer2 primer1 primer2 CSF1PO D5S818 D21S11 TH01 TPOX D13S317 D7S820 D16S539 D18S51 D8S1179 D3S1358 FGA VWA 13 CODIS Core STR Loci AMEL AMEL Sex-typing Position of Forensic STR Markers on Human Chromosomes Sample Detection CCD Panel Color Separation Ar+ LASER (488 nm) Fluorescence ABI Prism spectrograph Capillary or Gel Lane Size Separation Labeled DNA fragments (PCR products) Detection region Principles of Sample Separation and Detection Allelic Ladders PCR Product Size (bp) Sample #2 Sample #1 loci 8 11 14 All heterozygous alleles Crime Scene - Two Suspects Suspect 1 Suspect 2 Evidence D3 vWA FGA S1 14,15 17,18 23,24 S2 15,18 17,19 23.2,24 E 15,18 17,19 23.2,24 AMEL D3S1358 TH01 TPOX D2S1338 D19S433 FGA D21S11 D18S51 CSF1PO D16S539 D7S820 D13S317 D5S818 VWA D8S1179 1 integrated analysis vs. 16 separate runs Information is tied together with multiplex PCR and data analysis AmpFlSTR® Identifiler™ (Applied Biosystems) DNA Profile Frequency Locus allele value allele value frequency, 1 in D3S1358 16 0.2315 17 0.2118 10.20 VWA 17 0.2628 18 0.2219 8.57 FGA 21 0.1735 22 0.1888 15.26 D8S1179 12 0.1454 14 0.2015 17.07 D21S11 28 0.1658 30 0.2321 12.99 D18S51 14 0.1735 16 0.1071 26.91 D5S818 12 0.3539 13 0.1462 9.66 D13S317 11 0.3189 14 0.0357 43.92 D7S820 9 0.1478 43.28 D16S539 11 0.2723 13 0.1634 11.24 THO1 6 0.2266 18.83 TPOX 8 0.5443 3.35 CSF1PO 10 0.2537 15.09 The Random Match Probability for this profile in the FBI Caucasian population is 1 in 1.56 quadrillion (1015) AmpFlSTR® Identifiler™ (Applied Biosystems) AMEL D3 TH01 TPOX D2D19 FGA D21 D18 CSF D16 D7 D13 D5 VWAD8 PI = paternity index = 2(0.2315)(0.2118) = 0.0981 or 1 in 10.2 Combined DNA Index System Used for linking serial crimes and unsolved cases with repeat offenders Requires 13 core STR markers  For independent loci, the genotype frequencies can be combined through multiplication…  Profile Probability = Combined paternity index (CPI) = (P1)(P2)…(Pn) = 1 in a very large number… CODIS DNA Database Genetic Privacy Concerns  The DNA profile itself is neutral (and uninformative)— just a string of numbers like a Social Security Number  DNA markers used in forensics were selected to be neutral and are located away from or between genes rather than being part of gene products and therefore are not generally thought to be associated with any genetic disease  Concern is really with the DNA sample collected— would it be retained and used for any other type of testing? (e.g., Armed Forces DNA Repository can only be used for identifying combat casualties) PCR for DNA Profile Steps: 1. DNA sample analyzed for STR alleles present in population members 2. Analyze population frequency, how often combinations of alleles present 3. Population frequencies for each STR allele multiplied to estimate probability Combined Frequencies