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
◉ The 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 + PacManTaq Polymerase + PacMan

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

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

20. Scorpion Primers

21. The Process of Real Time PCR

22. One Step and Two Step Real Time PCR

23. Initial denaturation: 94oC for 15 minutes - 1 cycle, followed by
40 cycles of: Denaturation at 94.0oC for 15 seconds
Annealing at 55.8oC for 45 seconds
Extension at 72.0oC for 45 seconds
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

24. 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

25. Carrying Out Real Time PCR:

26. Absolute and Relative Quantification

27. Absolute Quantification

28. Relative Quantification- Comparative CT method
◉ Relative concentration of the gene of interest (GOI) in unknown samples is
compared to a calibrator, or control sample
◉ Examples:
1. Drug treated cell lines and untreated cell lines
2. Patient samples and healthy controls
◉ Differences in Ct value between an unknown sample and control are expressed
as fold-changes relative to the control sample (ΔCt)

29. 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., ΔΔCt = (CtTarget-CtACTB)Cases-(CtTarget-CtACTB)Controls
◉ Fold change = 2-ΔΔCt

30. Melting Curve

31. Melting Curve

32. Precautions

33. 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

34. 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

35. 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

36. Applications of Real Time PCR

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

38. Genotyping
High resolution melt curve analysis

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

41. THANKS!