Question

What are the three methods used for quantification of levels of expression in real-time PCR (qPCR)? describe how they work?

Answer 1

Real time Polymerase Chain Reaction is also known as quantitative polymerase chain reaction. It monitors the amplification of the target DNA in real time. Real time PCR can be used quantitatively ans semi-quantitatively. Semi-quantitative estimation of PCr takes place when the number of DNA molecules are present above/ below a certain threshold.

Quantitative estimation of PCR products is done by converting an mRNA into cDNA and using the former as a reaction substrate.The amount of the target gene transcript was compared to the transcript of the calibrator gene. This was done using comparative C_T method.

The other methods include Absolute quantification method and Relative quantification method. Absolute quantification relates the PCR to an input copy number to a calibration curve. It depends on conditions of identical amplification of the target DNA and calibration curve during the reaction. Relative quantification measures the relative change in the quantification of mRNA levels.The target genes and the housekeeping genes are compared and a calibration curve is not necessary in this regard.