By linking the PCR signal to a standard curve, Absolute quantification expresses the copy number of the transcript of target or control gene ( i.e.defining expression stages in absolute numbers of copies). Depending on the purpose of the study, two different methods, Absolute and Relative quantification, for evaluating data from quantitative PCR can be used. Considering all the prosperous applications of qPCR for gene copy number calculations, there are some aspects of the method, which raise concern regarding its accuracy.
There are main steps in determining copy number via qPCR, including: choosing the appropriate genomic region, choosing specific primers, practical validation of the primers, making accurate serial dilution of genomic DNA, and finally selecting an appropriate detecting chemicals. The logic behind qPCR is detecting the amount of the amplified genomic product in real time by means of various chemicals ( e.g.specific DNA binding dyes, hydrolysis probe, molecular beacon etc.), which leads to creation of a florescent sigmoid curve. Profound, and operational quantitative methods based on the PCR technique, including Competitive PCR and real-time PCR methods were improved to solve these difficulties. The technique quantifies the target genomic region and at the same time holds the detection potential.ĭespite the applicability of old quantitation methods ( i.e.Southern blotting) to measure transgene copies, the shortages appear while dealing with a large number of samples along with other obstacles like being time-consuming, involving substantial amounts of DNA from samples and using harmful radioisotopes in some instances. One of the demanding methods to quantify gene expression is Real-time Polymerase Chain Reaction.