Describe ways to improve PCR doubling efficiency.
Dr. Bronfin: I think doubling efficiency depends a lot on primer/probe specificity. Ideally melting temperature of reverse and forward primers should be within 5°C of each other. I know that some people suggest that adding additional MgCl2 may increase robustness, but I personally never saw any benefit from it.
Increasing extension time to 2–3 minutes may also help. Very often diluting your DNA prior to reaction in RNase/DNase free water can yield much higher efficiency, especially if your sample type is stool or cell lysate.
Dr. Mahajan: All our assays are optimized during the development phase to ensure reproducibility and maximum efficiency of PCR (i.e., doubling efficiency). We typically evaluate critical parameters such as primer and probe concentrations and reaction buffer conditions.
We use a standard optimization approach of running matrices of varying concentrations of reagents to find the optimal ratio that delivers maximal normalized fluorescence and minimal cycle threshold. We then test our optimized conditions on multiple standard curve preparations to ensure reproducibility.
Mr. Murakami: We have found that choice of master mix for the first cycling and including the correct Taq polymerase for the particular platform improves efficiency and productivity. To get earlier Ct value depends on the master mix. Multiple PCR is one of several ways to improve productivity. High-throughput instruments are another way to increase efficiency and throughput.
Dr. Palchevskiy: Quality RNA will provide good cDNA and consistent efficient amplification. Properly diluted sample is necessary as you do not want to exhaust your reagents or primers that are present in excess, except for house-keeping genes for multiplex qPCR reactions. A good primer/probe set that is highly sequence-specific to the gene (exon or exon-to-exon junction) of interest is helpful.
Another technique we employ is to re-purify the sample, if necessary, and perform a DNase digestion to prevent any source of DNA contamination. For SYBR, specificity of primers is absolutely essential, as well as checking the melting curve at the end of each run.
Mr. Papenfuss: Problems with PCR reaction efficiency can be attacked from several angles, and some or all of these may need to be explored. The quality of the primers and probes (if using a probe-based method) will affect everything. Be sure to use an established primer/probe design software package or website. Depending on the target, you may not be able to change your primers/probes much, if at all, but use the best ones you can.
Next, the master mix can affect reaction efficiency. If your PCR platform requires a specific master mix, by all means use it. If not, experiment with different mixes as they will each interact with your samples and reaction components differently. It is also possible to use any of a myriad of additives in the master mix, especially when dealing with difficult samples.
Lastly, the PCR cycling protocol directly affects reaction efficiency. Most systems are set up for the average sample, but if you have nonstandard samples, the average PCR cycling protocol may not be appropriate. Some systems are not adjustable, but if you are using a system that is, making changes to the temperature or duration of the various reaction steps can improve efficiency.