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qPCR Objective

To estimate the copy number of a target gene

About qPCR

Polymerase chain reaction (PCR) is a biochemical process that copies and amplifies DNA using a thermally stable DNA polymerase.

Real Time Quantitative PCR (qPCR) is a major development of PCR technology that enables reliable detection and measurement of products during each cycle of PCR reaction which are directly proportional to the amount of template prior to start of PCR process.

qPCR Principle

PCR is a technique for amplifying DNA. Generally, the PCR reaction consists of a series of temperature changes that are repeated 25 – 45 times.

These PCR cycles normally consist of three stages: the first, at around 95 °C, allows the separation of the nucleic acid’s double strands; the second, at a temperature of around 50-60 °C, allows the primer binding with the DNA template; the third, at between 68 – 72 °C, facilitates the polymerization carried out by the DNA polymerase.

This principle of amplification is utilized in real-time PCR also widely known as qPCR (quantitative PCR) but instead of looking at bands on a gel at the end of the reaction, the continuous reaction is monitored in “real-time”.

The reaction is placed in to a PCR machine that watches the reaction in real-time manner with a camera or fluorescence detector.

There are a lot of techniques out there that are used to allow the progress of a PCR process to be monitored but they all have one thing in common.

The DNA amplification to the generation of fluorescence which can be detected with a camera or sensor during each PCR cycle.

Hence, as the number of PCR copies increases during the reaction, so the fluorescence increases.

qPCR - research tweet 1

Adopted from BioRender

SYBR Green (or other intercalating dye)

SYBR® Green is by far the most commonly used intercalating dye. This dye operate via a simple mechanism.

The dye is fluorescent in its own but in the presence of double stranded DNA, the dye intercalates with the DNA double helix and thus alters the structure of the dye and causes it to fluoresce more.

An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity measured at each cycle.

Nomenclature Commonly Used in qPCR

Baseline is defined as PCR cycles in which a reporter fluorescent signal is accumulating but is beneath the limits of detection of the instrument.

Threshold is an arbitrary level of fluorescence chosen on the basis of baseline variability.

A signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (Ct) for a sample.

Threshold can be adjusted for each experiment so that it is in the region of exponential amplification across plots.

Ct is defined as the fractional PCR cycle number at which the reporter fluorescence is greater than the threshold.

The Ct is a basic principle of real time PCR and is essential component in producing accurate and reproducible data.

qPCR Applications

1. Quantitative mRNA expression studies.

2. DNA copy number measurements in genomic or viral DNAs.

3. Allelic discrimination assays or SNP genotyping.

4. Verification of microarray results.

5. Drug therapy efficacy.

6. DNA damage measurement.

Requirements for qPCR Assay

1. Genomic DNA (2-20 ng per reaction)

2. Primers for endogenous control and target gene

3. SYBR green

4. DNAse free Milli-Q water 5. Plasticware

6. Real time instrument

qPCR Procedure

1. Make a mastermix for 10 reactions.

ComponentsVol. per Reaction (ul)Vol. for 10 reaction (ul)
SYBR Green550
Forward Primer0.3 (300 nM-500 nM)3
Reverse Primer0.3 (300 nM-500 nM)3
cDNA1 (100 ng-100 fg)10
Water3.434
Total10100

2. Mix well and centrifuge briefly

3. Aliquote 9 μl of the mastermix in the wells of the 96-well plate

4. Add 1 μl of the DNA ( 50 ng/ μl) in each well and then seal the 96-well plate with

5. Setup the PCR reaction in the STEP ONE real-time PCR

6. Cycling condition 

7. Total number of cycle varies but usually 40.

StageTemperature (C) Min
Stage 1 (Denaturing)953:00
Stage 2 (Annealing)950:10
Stage 3 (Melt Curve)55-600:30
55-950:05

8. Introduce a hold at 4 ºC at the end of cycling if you need to store samples for some time (max. overnight) before the plate read.

9. Take reading in STEP ONE.

Formula for Calculating Relative Level of mRNA Expression

The relative level of mRNA Expression were calculated using the cycle threshold (Ct) method:

2−ΔΔCt = 2{ΔCt (treated samples) − ΔCt (untreated control)} where ΔCt = Ct (Gene of interest) − Ct (Endogenous Gene)

qPCR Citations

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