STEPS INVOLVED IN PERFORMING REAL-TIME PCR

Real-time PCR is a variation of the standard PCR technique that is commonly used to quantify DNA or RNA in a sample. Using sequence-specific primers, the number of copies of a particular DNA or RNA sequence can be determined. By measuring the amount of amplified product at each stage during the PCR cycle, quantification is possible.

If a particular sequence (DNA or RNA) is abundant in the sample, amplification is observed in earlier cycles; if the sequence is scarce, amplification is observed in later cycles. Quantification of amplified product is obtained using fluorescent probes or fluorescent DNA-binding dyes and real-time PCR instruments that measure fluorescence while performing the thermal cycling needed for the PCR reaction.

Real-time PCR steps
There are three major steps that make up each cycle in a real-time PCR reaction. Reactions are generally run for 40 cycles.

Denaturation

High-temperature incubation is used to “melt” double-stranded DNA into single strands and loosen secondary structure in single-stranded DNA. The highest temperature that the DNA polymerase can withstand is typically used (usually 95°C). The denaturation time can be increased if template GC
content is high.

Annealing

During annealing, complementary sequences have an opportunity to hybridize, so an appropriate temperature is used that is based on the calculated melting temperature (Tm) of the primers (typically 5°C below the Tm of the primer).

Extension

At 70–72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in real-time PCR is small, this step is often combined with the annealing step, using 60°C as the temperature.

Two-step qRT-PCR

Two-step quantitative reverse transcriptase PCR (qRT-PCR) starts with the reverse transcription of either total RNA or poly(A) RNA into cDNA using a reverse transcriptase (RT). This first-strand cDNA synthesis reaction can be primed using random primers, oligo(dT), or gene-specific primers (GSPs).

To give an equal representation of all targets in real-time PCR applications and to avoid the 3` bias ofoligo(dT) primers, many researchers use random primers or a mixture of oligo(dT) and random primers. The temperature used for cDNA synthesis depends on the RT enzyme chosen. After reverse transcription, approximately 10% of the cDNA is transferred to a separate tube for the real-time PCR reaction.

One-step qRT-PCR
One-step qRT-PCR combines the first-strand cDNA synthesis reaction and real-time PCR reaction in the same tube, simplifying reaction setup and reducing the possibility of contamination. Gene-specific primers (GSP) are required. This is because using oligo(dT) or random primers will generate nonspecific products in the one-step procedure and reduce the amount of product of interest.

Source

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