7.1 Quantitative/Real-Time PCR
(Note: For our lab we use Roche’s Universal Proble Library (preferred), and SYBR Green. The qPCR protocol is written for the UPL system.)
Quantitative PCR (qPCR) or Real Time PCR (rtPCR) is an older technique that has been largely replaced with Next Generation Sequencing (NGS) also known as High Throughput Sequencing (HTS) for some applications. Nevertheless, qPCR is still widely used for a multitude of experimental procedures including validation of NGS/HTS results. qPCR machines work the same as traditional thermocyclers, except they contain photodetectors for measuring fluorescent signals. During the DNA amplification process the fluorescence signal increases indicating an increase in DNA concentration. Using special software the qPCR machine can accurately calculate the starting concentration of the target gene. Check out this video to get a better understanding of qPCR. Check here for a more detailed explanation.
To actually start a qPCR experiment for measuring RNA expression, several things need to take place:
- Tissue collection
- RNA has to be isolated.
- cDNA libraries must be created from RNA samples.
- Primers and Probes have to be designed, ordered, resuspended, and diluted to the proper concentrations.
- The experiment has to be designed, and the workspace has to be prepared.
- A program has to be created on the LightCycler 96.