Getting the Most out of your Multiplex Assay
Multiplexing qPCR has many advantages. It can save setup time, reduce cost and minimize the amount of sample consumed in each experiment. However, there are some important considerations and additional work that must be done up front to make sure you get the most out of your multiplex assay, especially when validating one for the first time.
This video will take you step-by-step through the process of validating a qPCR multiplex assay to provide optimal results.
Let’s start from the beginning with the qPCR setup.
First, prepare a standard curve template. Start with a representative nucleic acid standard of a known concentration, and prepare a dilution series. Prepare at least six serial dilutions of 1:10 or 1:5. Always include appropriate controls, such as no template and no reverse transcriptase.
Then, combine 2X qPCR master mix, primers, probes, and water to prepare a sufficient volume of reaction mix for your standard curve and controls in triplicate plus a 10% excess for pipetting error for each condition. For a duplex reaction, make up one mix with assay 1, one mix with assay 2, and another mix with assays 1 and 2 combined. For multiplex reactions with greater than two targets, it is easiest to interpret results and identify problem primer/probe sets if each target is added sequentially to the multiplex reaction.
Now, add the reaction mix and template to the plate. Each template concentration should be tested in triplicate for each condition. Seal the plate and centrifuge briefly to collect the liquid at the bottom of the wells.
You also need to amplify reactions with the appropriate thermal cycling conditions. You should determine optimal denaturation and annealing/extension cycling conditions by designing primers with similar melting temperatures and testing singleplex reactions prior to this validation. A good starting point is the recommended standard cycling conditions for your qPCR reagent.
Next, you need to compare the performance of multiplex and singleplex reactions.
To validate your multiplex reaction, you must compare the Cq values, assay reproducibility, linearity, sensitivity and efficiency for single versus multiplex reactions with each primer/probe combination. If any of these five factors is significantly affected by multiplexing or if both singleplex and multiplex reactions do not perform as expected, further optimization or primer/probe changes may be necessary.
Finally, assess qPCR assay performance with this convenient tool that compares qPCR reagent used with your assay.
Quantifying expression of multiple target genes in the same qPCR reaction well makes sense. Following the multiplexing tips outlined in this video, you should be able to save setup time, reduce cost and minimize the amount of sample consumed in each experiment. And who doesn’t want that!