Biofiles 4.7

Protocol: Quantitative PCR for Determination

In recent years Quantitative PCR has reached a level of sensitivity, accuracy, and ease to support use as a routine assay for measuring gene level expression. The field of cancer research is currently validating a number of applications showing that qPCR can be a reliable tool for both researchers and clinicians concerning the behavior of tumors.1 Quantitative PCR is one of the molecular techniques providing the tools necessary to investigate tumor biology and to discover the genetic and epigenetic causes of cancer. qPCR is being used to analyze the biological differences between tumors that account for variations in morphology and clinical behavior. Quantitative PCR is playing an increasingly important role in clinical testing, providing information about gene expression, gene amplification or loss, and small alterations. It is also being used for detection and quantification of viral causes of cancer. qPCR has proven to be an extremely valuable diagnostic resource due to its objectivity, speed, versatility, and cost-effectiveness.2
Quantitative PCR uses the linearity of DNA amplification to determine absolute or relative amounts of a known sequence in a sample. By using a fluorescent reporter in the reaction, it is possible to measure DNA generation. In qPCR, DNA amplification is monitored at each cycle of PCR. When the DNA is in the log linear phase of amplification, the amount of fluorescence increases above the background. The point at which the fluorescence becomes measurable is called the Threshold cycle (CT) or crossing point. By using multiple dilutions of a known amount of standard DNA, a standard curve can be generated of log concentration against CT. The amount of DNA or cDNA in an unknown sample can then be calculated from its CT value. 

CT values for the lambda amplicon using SYBR® Green JumpStart™ Taq ReadyMix™. Quantitative PCR (qPCR) was performed on pBac-2cp. Initial template copy number was 106 and was diluted 10-fold in subsequent wells. Threshold cycles (CT) were determined using the ABI PRISM 7700 Sequence Detection software, and were found to be 15.304 (106), 18.848 (105), 22.883 (104), 26.208 (103), 29.821 (102), 33.398 (101), 37.038 (100), and 40 (0).
Issue QuickLinks