The conceptual and practical simplicity of quantitative real-time PCR (qPCR) has made it a choice tool for many molecular analysis applications. Because of its speed, sensitivity, and specificity, qPCR has been displacing conventional PCR in the vast majority of its applications. Some believe that it is only a matter of time before qPCR becomes a major player in diagnostics.
A large swath of publications reporting reverse transcriptase qPCR (RT-qPCR) data underscores a great interest in this technique. Yet there still exists a relative lack of consensus in how best to perform the RT-qPCR experiments.
“We can’t simply assume that RT-qPCR always accurately quantitates gene expression levels,” says Chaminda Salgado, head of CMC bioassay and genomics at NDA Analytics. “Each step of the procedure has technical challenges. Optimization and validation of each step is key.”
He adds that the “advancement of qPCR into the diagnostic realm will depend on standardization of at least four key technical components: Sample handling and assessment; RT strategy—enzyme selection and RT priming; normalization during analysis; and increase in hardware speed—for point-of-care applications.”
To date, standardization has been hampered by lack of sufficient experimental details in scientific reports. Frequently, qPCR publications omit critical analysis parameters and justification for reference gene selection, hindering critical evaluation of the quality of the results. To enable inter-laboratory comparisons, qPCR-based clinical diagnostic assays will require more stringent standardization.
Salgado emphasizes that primer design, reverse transcriptase selection, and choice of reference genes are vital underpinnings for the progression of RT-qPCR from a research technique to a market-ready in vitro diagnostic tool.
The first step toward this goal may be implementation of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. These call for reporting of minimal essential and desired information to ensure qPCR relevance, accuracy, correct interpretation, and repeatability. Properly implemented, the MIQE guidelines could help promote consistency among laboratories and potentially zero in on the methods that show potential utility for diagnostic applications.
“QPCR is a sensitive method, but many clinically important analyses require detection of minority events below qPCR current limits of sensitivity. Quantitation of rare mutations on the background of wild-type DNA is of prime importance in several fields of medicine,” says Mike Makrigiorgos, Ph.D., from the Dana-Farber Cancer Institute. “Specifically, subclonal mutations in cancer cells may determine if a particular tumor is resistant to chemotherapy. We envision that monitoring the quantity of these mutations during cancer treatment may inform management decisions for the remainder of the therapy or subsequent rounds of therapy.”
Dr. Makrigiorgos’ team focuses on development of techniques for identification of rare events and translating them into diagnostic applications. A few years ago, they developed COLD-PCR (co-amplification at lower denaturation temperature) to enrich samples with mutations irrespective of where they occur in DNA sequence. The technology takes advantage of a small but detectable difference in melting temperatures of mismatched sequences. Denaturing and re-annealing genomic DNA creates mismatched DNA duplexesFurther denaturation of these duplexes at critical denaturation temperatures preferentially releases the mismatched strands, which then can be amplified by PCR. Two consecutive rounds of COLD-PCR result in a 100-fold enrichment of mutations.
“Further enrichment with COLD-PCR was challenging due to polymerase errors and mis-priming events,” Dr. Makrigiorgos says. “We used the same scientific principle to develop an alternative enrichment method that does not involve enzymatic amplification.”
DISSECT (differential strand separation at critical temperature) also uses differential denaturation of DNA heteroduplexes. However, it avoids enzymatic steps being entirely based on repeated cycles of denaturation and hybridization on magnetic beads coated with wild-type target gene sequences. Wild-type DNA remains attached to the beads, while the mutant DNA is released and collected. Because the sequences are not altered during DISSECT, the method is compatible with downstream applications including qPCR and sequencing.
“The method is exceedingly simple, and is easy to automate, multiplex, and scale up,” explains Dr. Makrigiorgos. “DISSECT can simultaneously enrich diverse targets for multiple mutations in the same tube using a single denaturation temperature.” In a proof-of-principle study, three to four rounds of DISSECT produced up to 100- to 400-fold enrichment of mutations in three selected oncogenes.