Practical course on qRT-PCR

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Transcript of Practical course on qRT-PCR

  • 1. PRACTICAL COURSE ON QRT-PCR Course of Lectures for young scientists Molecular and Cellular Bases of Host-Microbes Interactions: Genomics and Sequencing 5th June 2014 Hovsep Ghazaryan
  • 2. GENE EXPRESSION ANALYZES 1. Isolation of RNA from cells / tissue / FFPE 2. Synthesis of cDNA (reverse transcription) 3. Quantitive Polymerase Chain Reaction 4. Statistical analyzes
  • 3. RNA ISOLATION METHODS 1. Guanidinium thyocyanate-phenol-chlorophorm method 2. Column purification A. RNA extraction from cells B. RNA extraction from whole blood C. RNA extraction from tissues D. RNA extraction from formalin-fixed, paraffin- embedded tissue samples
  • 4. REVERSE TRANSCRIPTION 65C for 5min, chill on ice Total RNA (0.1-5 g) 1 l Oligo dT primer 1 l Nuclease free water 9 l 37C for 60min, 70C for 5min MIX 11 l 5X reaction buffer 4 l RNase inhibitor (20 u/l) 1 l 10 mM dNTP mix 2 l Reverse transcriptase (20 u/l) 2 l
  • 5. PRIMER DESIGN Choose sense primer Choose antisense primer Choose probe Or use TaqMan assay
  • 6. PROBES Probes are short fragments of DNA of RNA. The probes are used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe. The probes are labeled with molecular marker of fluorescent or radioactive (in the past) molecules.
  • 7. WORKING SCHEME OF PROBES
  • 8. 1. GO TO QPCR.PROBEFINDER.COM
  • 9. 2. SELECT ORGANISM
  • 10. 3. SPECIFY YOUR TARGET
  • 11. 4. CHOOSE THE SEQUENCE
  • 12. 5. ENTER YOUR EMAIL ADDRESS AND PRESS DESIGN
  • 13. 7. PROBEFINDER WILL DESIGN OPTIMAL REAL-TIME PCR ASSAY
  • 14. TAQMAN ASSAYS
  • 15. CHOOSE HOUSEKEEPING GENE Housekeeping genes have constant expression under normal and patho-physologicial conditions For each type of cells / tissues there are different housekeeping genes Examples: GAPDH, HSP90, -actin, ACTB, PSMB1, PSMB2...
  • 16. CALLIBRATION Take reference cDNA for callibration with different concentrations: 10 g/ml, 5 g/ml, 2.5 g/ml, 1.25 g/ml
  • 17. QPCR 1 cycle 94C for 15 min, 40 cycles (94 C for 45 s, 60C for 30 s) dH2O 7.8 l 10X buffer 2.5 l 25 mM MgCl2 3.5 l 10 mM dNTPs 1 l Primer-L (10 pM/l) 2.25 l Primer-R (10 pM/l) 2.25 l Probe 0.5 l Taq polymerase 0.2 l
  • 18. QPCR RESULTS
  • 19. QPCR RESULTS
  • 20. QPCR RESULTS
  • 21. STATISTICAL ANALYSIS Check normal distribution with Kolmogorov-Smirnov test If passes normality test, use Students T-test For nonparametric distribution use Mann-Whitney U-test
  • 22. STATISTICAL ANALYSIS