Ian Kavanagh, Ph.D., R&D manager of genomics at Thermo Fisher Scientific, also had advice on how to minimize variance. He said a few quick and simple modifications to one’s protocol can minimize many common sources of variance.
“Many things can go wrong with qPCR and there are a lot of choices labs must make to minimize user variance. A user may have to contend with DNA contamination, which can cause false positives since PCR cannot distinguish between cDNA and genomic DNA. An easy fix is to include shrimp nuclease that destroys double-stranded DNA during the reverse-transcription step, but which is inactivated by high temperatures at the conclusion of that step.”
Another recommendation is to use white qPCR plastics instead of the traditional clear plastics. “Our studies found that use of white plastics can greatly enhance sensitivity, especially at lower limits of detection,” Dr. Kavanagh noted. “Some of the resistance to adopting white plates is that samples may not be as visible in the wells. We recommend use of an inert blue dye added to the reaction and have launched a product line called Thermo Scientific ABsolute Blue that contains this dye in the mix.”
Speed is usually a desirable quality in any scientific experiment. Fast qPCR is a method that reduces the time of the protocol. According to Dr. Kavanagh, “a common misconception is that fast qPCR requires specialized instrumentation. In reality, you can use standard instrumentation with optimized reagents and cycling conditions. The key to success is carefully designing the assay to minimize the secondary structure within the amplicon and to restrict the length to less than 150 base pairs.
“There will always be areas where variability can creep into protocols. While some are hard to get around, others have less complex solutions such as with DNA contamination and fast cycling conditions. Scientists can drastically improve reproducibility of data and increase the confidence in their results by making a few simple changes. Ultimately, that will save you both time and money,” Dr. Kavanagh concluded.
Solving Multiplexing Issues
Multiplex, real-time PCR enables the amplification of two or more targets in a single reaction. “It’s increasingly being accepted as the technology of choice when needing to simultaneously analyze expression of one or more genes of interest with reference genes as well as for pathogen detection,” said Mark Richards, marketing manager at Qiagen.
Richards noted that researchers still face many challenges when trying to optimize multiplex qPCR. “Multiplexing can be tedious and time consuming because of the need to establish optimization procedures such as adjusting enzyme, primer, DNA, and magnesium concentrations in order to provide the best signal-to-noise ratio. Even after this extensive work, many cases still prove disappointing.”
Qiagen has developed a number of technologies for multiplex PCR that eliminate the need for optimization. “The multiplex PCR technology uses a highly stringent hot-start enzyme, an ammonium containing buffer, and a multiplex PCR-enhancing synthetic factor MP that eliminates the need for optimization because it supports macromolecular crowding. That is, it allows efficient annealing of multiple primers under identical cycling conditions. Scientists have shown that they can detect up to seven targets with this technology.”
“With this system, primer concentrations do not have to be optimized. One can use the same concentration of each. Of course, the primers must be suitable and optimized for the sequence, just as in any singleplex assay,” Annette Tietze, Ph.D., senior global product manager for qPCR, added.