Gene Expression Service—Quantitative PCR (qPCR)

Introduction

Templates
Once you have established your conditions, it is important to titrate your cDNA template such that you use the minimum required to obtain Cq values between 10 and 30 cycles. In addition, once your conditions are established, you should make a standard curve of your primers utilizing different concentrations of template. The slope is a measure of efficiency. One hundred percent efficiency would mean that there is a perfect doubling of product at each cycle. Less than 100% efficiency should be accounted for in the final results. The instruments will calculate the efficiency for you, so this is not difficult to do and need only be done once or twice if your efficiencies are near 100%.
Primer Design
Primers should be designed such that the products are typically between 100 bp and 200 bp, although in practice much longer products may also work well. There is nothing special about qPCR primers. The key is to be sure they do not form dimers, secondary structures or are non-specific. If you are unsure of the specificity, test them first using conventional PCR and examine the product on a gel. A handy set of online tools for such analyses are available at the website for Integrated DNA technologies (IDT).

Applications

1. At a linear phase of amplification, the detection of PCR products (and their corresponding mRNAs) is limited only by the quantity of template. Thus, qPCR provides a sensitive and accurate determination of relative or absolute mRNA abundance and gene expression compared to RT PCR, which measures mRNA abundance at a fixed time point.
2. qPCR has many applications including absolute and relative gene expression, copy number, miRNA, allelic discrimination, detection of viral or bacterial genomes, and 16S ribosomal genes for metagenomics.

CD Genomics provides gene expression services based on Quantitative PCR (qPCR). For more information, please feel free to contact us.