April 1, 2009 (Vol. 29, No. 7)

Reducing Cycling Time and Improving Accuracy Are among the Benefits

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

Also presenting at the meeting was Natasha Paul, Ph.D., senior staff scientist at TriLink BioTechnologies.

Thermo Fisher Scientific reports that it has achieved dramatically reduced qPCR dwell times with no drop-off in amplification efficiency (Cp values).

Reducing Nonspecific Amplification

PCR is a widely accepted thermal cycling process that provides approximately 106-fold amplification of a nucleic acid target of interest. Dr. Paul said that it is increasingly used in high-stringency applications such as molecular diagnostics where the requirement is to detect down to as few copies as possible without false positives or false negatives. 

“The process can be plagued by competing off-target amplicon formation such as mispriming and primer dimer formation,” she noted. TriLink’s Hot Start activation is a strategy to reduce nonspecific amplification during the less stringent set-up stages of PCR, she explained.

The goal of Hot Start technologies is to block primer extension at lower temperatures, but to allow for reactivation of the process at higher temperatures. TriLink has developed two approaches to Hot Start activation in PCR that employ chemical modifications to two widely underutilized components of the reaction—the primers and the dNTPs.

The modified primer technology employs a thermolabile blocker, in the form of 4-oxo-tetradecyl, to the 3´ phosphodiester backbone of the primer molecule. The blocker is released at 94ºC, leaving a normal, unmodified primer that can be extended. The CleanAmp™ Turbo Primer features one blocker moiety while the Precision Primer features a double substitution of the group.

CleanAmp Turbo Primers are used in fast cycling (two-step protocols) for multiplexed PCR of DNA templates. CleanAmp Precision Primers are applied in standard cycling (three-step protocols) and in one-step reverse transcription PCR.

Dr. Paul noted that this modified primer technology is of particular utility in one step RT-PCR set-ups, where the lower temperature reverse-transcription step is accomplished with a poly-dT reverse transcription primer and also includes PCR primers. These primers are blocked from extension by CleanAmp Precision modifications until the tube is heated up to PCR temperatures. 

According to Dr. Paul, CleanAmp  Precision PCR Primers enhance results in multiplex one-step RT-PCR when compared to unmodified PCR primers, which are generally available throughout the reaction. Dr. Paul said that results from tests in liver RNA indicate that the CleanAmp Precision Primer modifications show promise for relative RNA quantification in real-time one-step RT-PCR.

Hot Start activation works in largely the same way with TriLink’s modified deoxyribonucleotide triphosphate (dNTP) technology. The thermolabile protective group in this case is a tetrahydrofuranyl modification to the 3´ hydroxyl, which has been shown to result in improved PCR performance for several targets.

In a demonstration of increased sensitivity, the use of CleanAmp dNTPs detected DNA at 5,000, 500, 50, and 5 copy levels, unmodified dNTPs were effective at only the 5,000 and 500 copy levels. CleanAmp dNTPs were also found to enhance PCR specificity in comparison with unmodified dNTPs when used in combination with CleanAmp Precision Primers, Dr. Paul said, essentially eliminating detectable primer-dimer formation, indicating that the use of CleanAmp dNTP and CleanAmp primers together provide a synergistic effect.

TaqMan Chemistries

Pointing out that miRNA can lead to new areas of biomolecular control for use in both therapeutics and diagnostics, BioTrove’s CTO Colin Brenan, Ph.D., talked about his company’s newly launched OpenArray® DLP Real-Time qPCR platform, providing the first fully licensed high-density qPCR platform for use with customers’ TaqMan® chemistries. The platform delivers new efficiencies to support its use as a discovery tool, capable of evaluating entire libraries of miRNA (with three specimens per plate and three plates per run or nine specimens in one shot) and quantitatively determining the change in microarray abundance within a two-hour cycle time.

In addition, Dr. Brenan noted, microarrays lack precision and sensitivity. He cited a study conducted with Beth Israel Hospital and the Laboratory of Innovative Translational Technologies at Harvard  where kinase gene expression in prostate cancer specimens that determined 40% of kinase genes in the human kinome, but were not detected in microarrays, were found to be differentially expressed in OpenArray. A new study is being conducted to validate these findings.

The OpenArray DLP Real-Time qPCR platform provides a straightforward method for profiling miRNAs, according to Dr. Brenan, by detecting pathogens, validating microarray results, and performing expression-based biomarker screens. The combination of TaqMan assays with OpenArray Real-Time qPCR genomic analysis enables better use of staff, reagent supply, and resources, and boosts productivity, Dr. Brenan noted.

“The OpenArray DLP platform enables researchers to complete projects in days instead of weeks, providing unprecedented gene coverage and sample throughput in a unique parallel-array format using TaqMan chemistry.”

Last year, Applied Biosystems (now Life Technologies) and BioTrove launched the TaqMan OpenArray Genotyping System for PCR-based SNP analysis, which is exclusively marketed by Life Technologies. Targeted markets for the new capability include pharmaceutical development to validate microarray hits at lower cost and with higher productivity, and human diagnostics where multiple pathogens can be consolidated onto a single OpenArray plate and analyze multiple specimens simultaneously.

miRNA has been identified as an important regulator of gene expression, Jason Halsey, vp of molecular biology systems R&D for Life Technologies, told the conference. Dysregulation of miRNA is often associated with diseases like cancer. 

Approximately 695 human miRNA genes have been identified to date, he noted, and many researchers believe that hundreds of additional human miRNAs are yet to be discovered. Conventional cloning and Sanger sequencing methods may not achieve this increasingly difficult task as evidence suggests that unknown miRNAs may be present in only a specific cell type, at a particular developmental stage, or at relatively low expression levels.

The Life Technologies team used an Applied Biosystems workflow to find and validate new miRNAs. The discovery of new miRNAs was performed on the SOLiD™ platform by sequencing small RNA species between 18 and 40 nucleotides found in 10 human tissue samples (placenta, heart, brain, liver, testes, spleen, kidney, thymus, lung, and ovary).  The validation of newly found miRNAs  was performed by utilizing custom-designed small RNA TaqMan assays.

The new workflow allows users to purify small RNA using the Ambion mirVana miRNA Isolation kit in conjunction with the SOLiD Small RNA Expression Kit to make sequencing ready libraries from 1 to 500 ng of small RNA, Halsey said. The method is highly reproducible across samples, operators and labs (R square >0.98), he added. Using this method on placenta, human tissue yielded over 85 million mappable sequencing tags—51% of all reads mapped to Sanger mirBase, leaving 37 million matched to the genome only. 

Sequencing data of known miRNA showed unique properties of isomiRs. For many miRNA the most abundant versions started from a different location than the Sanger reference sequence, and Dicer does not randomly cleave mature miRNAs but favors isomiRs with A or U at 5´ terminal.

“We have developed an approach using a commercial kit with which we prepare libraries via a five-step process,” Halsey related. Using 1 ng to 500 ng of 18–40 bp nucleotide samples, small RNA are hybridized to an adapter that is ligated and reverse transcribed. RNase digestion leaves single-stranded cDNA, which is then gel purified and ready for SOLiD library preparation. The adaptor can also include a bar code so users can sequence up to 16 samples simultaneously on one slide of the SOLiD platform. 

This method enables the simultaneous and directional ligation of adaptors to the ends of RNA as the first step in the construction of small RNA libraries suitable for the SOLiD platform, according to Halsey. “We constructed and sequenced ten small RNA libraries from ten different human tissues, generating approximately 260 million total sequencing tags.” Analysis of this data demonstrated detection of up to six logs of dynamic range and correlation to real-time PCR, indicating its capability of deep sequencing as well as profiling small noncoding RNAs. 

“Using an in silico support vector machine algorithm, we developed a method to predict miRNA-like sequences present in the various tissue samples and found a few hundred potential miRNAs in the placenta sample,” Halsey stated. Forty custom TaqMan small RNA assays were designed and tested against eight tissue samples, validating 33. These results demonstrate a simplified, quantitative, and sensitive methodology for both profiling and discovery of small noncoding RNA targets including miRNAs, using the SOLiD system, Halsey added. Furthermore, custom TaqMan small RNA assays can be used for follow-up screening and validation to identify miRNAs related to specific diseases.

“I think the take-home message is: although tremendous work has been done to discover miRNA, this workflow will allow people to accelerate discovery,” Halsey concluded.

According to TriLink BioTechnologies, its CleanAmp Precision PCR Primers enhance results in multiplex, one-step RT-PCR when compared to unmodified PCR primers.

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