Scientists convened in Munich last month for the “International qPCR Symposium” to discuss what the event’s organizers described as “the most powerful analytical technology ever developed in the life sciences area—the quantitative real-time polymerase chain reaction (qPCR).”
Among the more than 50 speakers was Thermo Fisher Scientific’s Ian Kavanagh, Ph.D., research and development manager, genomics, who discussed how it is possible to reduce qPCR cycling time, yet maintain robust data in the vast majority of instances. In his talk, Dr. Kavanagh examined workflows for gene modulation that utilize the delivery of functional siRNA, determine target gene knockdown via qPCR, and then assess the biological phenotype created by silencing the targeted gene.
Dr. Kavanagh shared his insights on what can go wrong in the qPCR process. Among the problem areas he cited was pipetting, nucleic acid isolation, RNA degradation, loss of sample, DNA contamination, RT efficiency, and data normalization. His point was that, although qPCR can look daunting, it is possible to generate robust data across a template range of nine orders of magnitude in less than two hours, comprising a reaction set-up time of 30 minutes, run-time or “fast qPCR” of 60 minutes, and data analysis of 30 minutes.
To define what he means by “fast qPCR” Dr. Kavanagh referred to the standard PCR-cycling protocol for a probe chemistry with a hot start at 95ºC for 2 to 15 minutes, followed by amplification for 35 to 45 cycles at 95ºC for 10 seconds and 60ºC for 60 seconds. “Can these dwell times be reduced?” he asked.
What followed, answered the question with an emphatic “yes”. Dr. Kavanagh showed dramatically reduced dwell times with no drop-off in Cp values for RNase P 80 bp target amplified from human genomic DNA and ApoB 74, respectively. Similar results were demonstrated with human albumin 104 and GAPDH 226.
There are reasons that amplification efficiency is sometimes reduced in fast cycling, Dr. Kavanagh said, such as primer annealing rate, GC%, secondary structure, enzyme activity, and amplicon length. Secondary structure and amplicon length are closely related, he noted, and are most likely the biggest culprits. But “the majority of researchers work with short amplicons and should be able to use fast cycling,” he stated, while perhaps 90% of qPCR assays in common, use yielding amplicons of less than 200 bp. Indeed, a list of 100 gene sequences input into Primer 3 Plus returned no assays with an amplicon larger than 200 bp.
What this can mean in terms of time savings was demonstrated by evaluating one standard and two fast qPCR instruments with three different peak cycle ramp rates. Protocol times in minutes could be decreased from approximately 125 to 60, 105 to 55, and 105 to 50, respectively, which represent almost a 50% reduction in cycling time.
Dr. Kavanagh concluded that fast qPCR cycling could work for the majority of genes with <150 bp amplicon length, without any reduction in sensitivity and maintaining the reproducibility of the data. In terms of future directions, he would like to see a reduction in set-up steps, and speedup the analysis steps by using single curve efficiency calculations and simple relative quantification calculations.
Also presenting at the meeting was Natasha Paul, Ph.D., senior staff scientist at TriLink BioTechnologies.