What is required in developing multiplex Real-Time PCR assays?
Developing multiplex Real-Time PCR assays can be difficult and time-consuming. As the reaction complexity increases, significant optimization may be required to generate reliable data. It can be a challenge to develop multiplex assays that amplify all targets with equal efficiency. When developing multiplex Real-Time PCR assays, you need to consider primer design, the relative expression levels of target sequences, and master mix / reagent conditions. Use the same design criteria for each primer/probe set and screen all sequences against each other to determine any potential primer-dimer formation. In addition, perform a BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine primer specificity. If the expression levels of the target sequences are significantly different, the most abundant target will be preferentially amplified and deplete all the reaction components, compromising amplification of the less abundant targets. One way to address this issue is to limit the p
