It’s been over a quarter of a century since the discovery of PCR, followed by the development of quantitative PCR (qPCR), also known as real-time PCR, the breakthrough that enables quantification of target molecules in the initial sample. qPCR is a multistep assay and each step must be optimized to deliver accurate results. Recent technology advances have addressed speed, throughput, convenience and affordability, yet obtaining reliable results remains a challenge.
“People think that because qPCR is such a superb technique, it must be simple,” explains Emir Hodzic, PhD, Director of Real-time PCR at the University of California at Davis Real-Time PCR Research and Diagnostic Core Lab. But there are multiple crucial steps and details that have cascading effects. If any part of the process is missing or performed incorrectly, the data will suffer. There are a dozen or so parts of the qPCR process that are ripe for optimization. These tips should get you off to a good start.
Sample Preparation and Quality Control
In qPCR, the adage “garbage in – garbage out” is really true, according to a PhD who is currently working in the area of DNA isolation and purification. “If your sample prep is not properly done, you may have carry-over inhibitors and proteins that will cause quantification problems, create ‘false negatives’ and inhibit downstream applications,” she continued.
In addition, the integrity of the starting material itself needs to be of the highest quality. Sheared genomic DNA or degraded RNA can also generate false negatives or sub-optimal results. To ensure high quality starting material, perform nucleic acid extractions carefully and always evaluate the quality of your DNA or RNA prior to running your qPCR assay. You can perform quality control checks using an array of tools, from traditional Northern blotting or agarose gel electrophoresis to analysis utilizing automated microchip electrophoresis.
Normalization strategy
Too many gene expression studies have been published in which the use of only one reference gene has brought all of the data into question. Normalization against just one gene can no longer “pass muster.” Researchers need to take the time to complete and document a more thorough normalization process, even though it adds to the total project time.
According to OpenWetWare, a hub for biological collaboration, an ideal reference gene should be expressed at the same level in all cells and the level of expression should approximate the level at which your gene of interest is expressed. Any gene will show altered expression under some set of conditions; this is why it is important to select a set of reference genes that match up well against the conditions you are studying, and will provide you with a consistent point of reference. Normalizing your target gene expression to 3 or so qualified reference genes will strengthen the integrity of your data and give you a much clearer understanding of the variations in expression that you observe.
Primer and probe design
The last thing you want to do is design a primer pair or probe that isn’t as discriminating as you thought it was and as a result winds up binding to alternate sites. Make sure to always check your primer and probe sequence against the NCBI’s primer BLAST database. In addition to validating specificity, there are numerous other parameters to consider, including melting temperature, annealing temperature, the likelihood of secondary structure, primer/probe length, amplicon length and more. The surest way to factor all of these parameters into your primer and probe design is to use a respected primer design tool. There are numerous such tools available, from free online resources to software packages that can be purchased from vendors specializing in this area. One thing is for sure, primer and probe design are essential to generating the most robust and specific assay possible.
Consider the MIQE Guidelines
Concerns over assay quality control and standardization have been an impediment to the expanded use of qPCR. One set of guidelines that has gained traction is the “Minimum Information for Publication of Quantitative Real-Time PCR (MIQE) Guidelines.” MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments (1). The guidelines include a checklist to accompany the submission of a manuscript to a journal.
A powerful technique
qPCR is most certainly a powerful and superb technique, but assay design and execution are not as simple as the traditional PCR which preceded it. Attention to each aspect of designing and performing your qPCR assay will help you deliver truly powerful and superb results.
Reference:
(1) Bustin SA et al., “The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.” Clin Chem. 2009 Apr;55(4):611-22.
The image at the top of this page is from Agilent Technologies.