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

New qPCR Workflows Accelerate Discovery

Reducing Cycling Time and Improving Accuracy Are among the Benefits

  • TaqMan Chemistries

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

    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.

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