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
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
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
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
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
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
absorbs blue light (λmax = 497 nm) and emits green light (λmax = 520 nm)
Several different fluorophores (e.g. 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescein, acronym: TET) and quenchers (e.g. tetramethylrhodamine, acronym: TAMRA) are available
Concentration is measured by A260 and converted to the number of copies using the molecular weight of the DNA or RNA.