Real-Time or Quantitative PCR

Real-time PCR is employed for the estimation of differential mRNA expression profile of genes. It is the quantitative expression of a series of genes from cDNA. The basic principle behind the technique is the progressive recording of PCR-based DNA amplification by continuous recording change in florescence. Real-time PCR primers are designed by aligning gene sequences of several mammals. QPCR technique may involve either Sybergreen chemistry or Taqman probing. The former is simpler to analyze, while later gives more accurate results. QPCR study is conducted from cDNA derived from different sources under study. Equal amount of RNA (quantified by Qubit fluorometer, Invitrogen), wherever applicable, are used for cDNA preparation (Superscript III cDNA synthesis kit; Invitrogen). All qRT-PCR reactions are conducted on ABI 7500 fast system. Each reaction consisted of 2 μL cDNA template, 5 μL of 2× SYBR Green PCR Master Mix, 0.25 μL each of forward and reverse primers (10 pmol/μL), and nuclease-free water for a final volume of 10 μL. Each sample is run in duplicate or triplicate. Analysis of real-time PCR (qRT-PCR) is performed by delta-delta-Ct (ΔΔCt) method . Expression level of mRNA is detected by 2−ΔΔCt