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Real-Time PCR Principle and Procedure

In this Real-Time PCR principle and procedure post we have briefly explained about Real-Time PCR Test, principle, reagent kit, and Real-Time PCR Test procedure.

Real-Time PCR Test

Reverse transcriptase PCR or Real-Time PCR Test was developed to amplify RNA targets (RNA viruses such as HIV, HCV, and influenza are key examples). Essentially, the method entails an initial step of transcribing a portion of the RNA genome into complementary DNA (cDNA) which is then amplified through PCR. 

PCR depends on the Taq Polymerase enzyme; RNA is not an efficient substrate for this enzyme. This is why the target of interest (if present) is first transcribed into complementary DNA (cDNA), which can then be amplified.

Principle

Real-Time PCR Test involves the conversion of mRNA of gene of interest present in the total RNA into cDNA and then amplifies a specific region of the cDNA. This enzyme reverse transcriptase catalyzes the conversion of mRNA into cDNA. This enzyme is isolated from retroviruses such as murine moloney leukemia virus (MMLV) and avian myeloblastosis virus (AMV). Reverse transcriptase polymerase chain reactions were done using a single step kit in which the reverse transcription reaction and the amplification can be carried out in a single vial.

RT-PCR Reagents

Enzyme mix: Enzyme mix containing 20 mM Tris HCl, 100 mM KCl,1 mM dithiothreitol (DTT), 0.1mM EDTA, 0.5%(V/V) Stabilizer; pH 9.0.

5x RT-PCR master mix: 5x RT-PCR master mix containing Tris HCl, KCl, (NH4)2SO4, 12.5 mM MgCl2, DTT, final pH adjusted to 8.7.

dNTP Mix: dNTP Mix contains premixed aqueous solutions of dATP, dCTP, dGTP and dTTP, each at a final concentration of 10 mM.

RNase free water: RNase-Free Water is prepared with a proprietary process, which yields
RNase-free, deionized water without the use of chemical additives, such as diethylpyrocarbonate (DEPC).

Oligonucleotide primers: For amplification of the individual gene, a 18-21 base primer needs to be identified and synthesized (available commercially).

Procedure

Reverse Transcription is carried out with the SuperScript First-Strand Synthesis System for Real-Time PCR Test. The following procedure is based on Invitrogen’s protocol.

Reverse transcriptase PCR (RT-PCR)

Prepare the following RNA/primer mixture in each tube Reverse Transcription (Real-Time PCR Test) cDNA synthesis

Total RNA

5µg

Random hexamers (50 ng/ml)

3µl

10 mM dNTP mix

3µl

DEPC H2O

Made up to 10µl

Incubate the samples at 65°C for 5 min and then on ice for at least 1 min. Prepare reaction master mixture. For each reaction:

10x RT buffer

2µl

25 mM MgCl2

4µl

0.1 M DTT

2µl

RNAaseOUT

1µl

Add the reaction mixture to the RNA/primer mixture, mix briefly, and then place at room temperature for 2 min. Add 1 ml (50 units) of SuperScript II RT to each tube, mix and incubate at 25°C for 10 min. Incubate the tubes at 42°C for 50 min, heat inactivate at 70°C for 15 min, and then chill on ice. Add 1 ml RNase H and incubate at 37°C for 20 min. Store the 1st strand cDNA at -20°C until use for Real-Time PCR Test.

  1. Reverse transcription (RT) : 50°C for 30 min
  2. Termination of RT: 94°C for 2 min
  3. Denaturation: 94°C for 90 sec
  4. Annealing: 55°C for 90 sec (30 cycles)
  5. Extension: 72°C for 90 sec
  6. Final extension: 72°C for 10 min and Hold at 4°C

Further Readings

Reference