REAL TIME PCR
Real-time PCR is an extremely sensitive and reproducible method to measure levels of gene expression in an organism. Indirectly, the expression of particular genes may be assessed with microarray technology, which can provide a rough measure of the cellular concentration of different messenger RNAs; often thousands at a time. While the name of this type of assessment is actually a misnomer, it is often referred to as expression profiling. The expression of many genes is known to be regulated after transcription, so an increase in mRNA concentration need not always increase expression. A more sensitive and more accurate method of relative gene expression measurement is real-time polymerase chain reaction. With a carefully constructed standard curve, it can even produce an absolute measurement such as in number of copies of mRNA per nanolitre of homogenized tissue, or in number of copies of mRNA per total poly-adenosine RNA. Real-time PCR is a natural follow-on to microarray analysis. The genes that have been determined to be differentially regulated are subjected to RT-PCR to look at fold changes and to evaluate the statistical significance of the change.
EcoArray’s thorough processing incorporates the following steps:
- Design custom primers and probe sets using state of the art software and design parameters.
- Optimize the custom primers and probe sets. Each primer set is validated for specificity and efficiency by running dissociation and standard curves, respectively. Standard curves include data from a minimum of four serially diluted cDNA samples. The efficiency of amplification will be >95% for each primer set including 18S rRNA, which is used as a normalizing gene. If there is reason to suspect 18S does fluctuate, another gene can be used for normalization.
- Set up and run the real-time PCR reactions. High quality total RNA samples are DNase-treated, reverse transcribed to single-strand cDNA, and run in duplicate alongside no-RT controls.
- Analyze the data. The averaged value is normalized to measured 18S rRNA values for each sample. To evaluate the results a ∆∆Ct method of analysis is used to compare changes in gene expression between controls and treated samples.
Typical Real-time PCR experiment: Design real-time PCR primers for 5 genes. Run 40 samples (DNase-treated total RNA supplied) against each one plus 18S.