The method of choice for analysis of the gene expression levels of a small number or a panel of genes is Real-Time PCR. Real-Time PCR, also referred to as Quantitative PCR (or qPCR), was developed as a precise, efficient and rapid method for nucleic acid detection. Far different from the former end-point method of standard PCR techniques, Real-Time PCR can be quantitative because the PCR product is detected using fluorescent dyes in real time. It has the characteristics of sensitivity, specificity and a wide dynamic range and is thus often used to validate other techniques such as microarray or RNAseq on smaller gene panels.
Two common methods for the detection of PCR products in real-time PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence.
A variety of platforms now exist for any laboratory to gain access to qPCR. The choice of platform depends on the throughput and quality of result desired.
Roche LightCycler - Unleash the Potential of Real-Time PCRThe LightCycler® 480 System is a plate-based, highly adaptable and versatile real-time PCR system for gene expression analysis, SNP genotyping, and mutation scanning via high resolution melting (HRM). The modern instrument design, outstanding technical features, and comprehensive software make the LightCycler® 480 System your platform of choice for high speed and accuracy in all current real-time PCR applications.
