Real Time PCR

A basic outline of Real Time PCR covering detection chemistry, quantification techniques, recent advances, pitfalls etc.
Prepared in August 2016

1. Real Time PCR Ashikh Seethy Senior Resident and PhD Scholar Dept of Biochemistry AIIMS- New Delhi

2. Studying Gene Expression RNA extraction and quantification cDNA synthesis and confirmation of synthesis End Point PCR or Real Time PCR

3. Overview: What is Real Time PCR? Why Real Time PCR? Chemistries used in Real Time PCR T he process of Real Time PCR Absolute and relative quantification Melting curve Precautions Applications

4. What is Real Time PCR? Why Real Time PCR?

5. Phases of PCR

6. Problems with detection in the plateau phase of PCR

7. Problems with end-point detection

8. Problems with end-point detection Poor precision Low sensitivity Low resolution Non - automated Size-based discrimination only Ethidium bromide for staining

9. Alternative? Real Time PCR Real Time PCR uses fluorescence detecting thermocyclers to amplify specific nucleic acid sequences and measure their concentrations simultaneously

10. Chemistries in Real Time PCR

11. Chemistries in Real Time PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers

12. Chemistries in Real Time PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers

13. SYBR Green I

14. SYBR Green I Limitations: Detection of non-specific ds reaction products Increased background or false positives Inhibition of the PCR reaction Other dyes: SYTO 9 Evagreen

15. Chemistries in Real Time PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers

16. Taq Polymerase + PacMan Taq Polymerase + Pac Man

17. Chemistries in Real Time PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers

18. Chemistries in Real Time PCR DNA Binding Dyes Taqman probes Molecular Beacons Scorpion Primers

19. Scorpion Primers

20. The Process of Real Time PCR

21. One Step and Two Step Real Time PCR

22. Initial denaturation: 94 o C for 15 minutes - 1 cycle, followed by 40 cycles of: Denaturation at 94.0 o C for 15 seconds Annealing at 55.8 o C for 45 seconds Extension at 72.0 o C for 45 second s Sl No. Reagent Quantity/Reaction 1. cDNA 2 µL 2. Maxima SYBR Green/ ROX qPCR Master Mix 6 µL 3. Forward primer – 25 pmol /µL 0.3 µL 4. Reverse primer – 25 pmol /µL 0.3 µL 5. Nuclease free water 12 µL

23. Controls in Real Time PCR: Non-Template Control/ Negative Control Minus RT control detects DNA contamination No amplification control Positive control- exogenous/ endogenous Internal/ Normalisation control Passive reference dye

24. Carrying Out Real Time PCR:

25. Absolute and Relative Quantification

26. Absolute Quantification

27. Relative Quantification- Comparative C T method Relative concentration of the gene of interest (GOI) in unknown samples is compared to a calibrator, or control sample Examples: Drug treated cell lines and untreated cell lines Patient samples and healthy controls Differences in C t value between an unknown sample and control are expressed as fold-changes relative to the control sample ( Δ C t )

28. Relative Quantification- Comparative CT method To control the differences in RNA isolation and in the efficiency of the reverse transcription from sample to sample and experiment to experiment ↓ Normalization with reference gene, typically a gene whose expression is constant in both the control and experimental samples like 18S rRNA, GAPDH and ACTB . i.e., ΔΔ C t = ( Ct Target -Ct ACTB ) Case s -( Ct Targe t -Ct ACTB ) Controls Fold change = 2 - ΔΔ Ct

29. Melting Curve

30. Melting Curve

31. Precautions

32. Precautions: Product length: 70– 150 bp for probe-based chemistries 100– 300 bp for SYBR Green Primers incorporating exon-exon junctions RNA extraction and quantification cDNA synthesis and confirmation of synthesis Real Time PCR

33. Precautions: Avoid primer dimerization and misfolding : GC clamps Inverted repeats Melting Curve Analysis Controls RNA extraction and quantification cDNA synthesis and confirmation of synthesis Real Time PCR

34. Precautions: SYBR Green I is light sensitive Avoid marking on PCR tubes Prepare a cocktail Pipetting RNA extraction and quantification cDNA synthesis and confirmation of synthesis Real Time PCR

35. Applications of Real Time PCR

36. Applications Viral Quantitation Quantitation of Gene Expression Array Verification Pathogen detection Drug Therapy Efficacy Genotyping

37. Genotyping High resolution melt curve analysis

38. Genotyping Minor Groove Binding Taqman Probes Locked Nucleic Acid Probes

39. THANKS!