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May 1, 2011 (Vol. 31, No. 9)

qPCR Evolving to Meet Emerging Needs

Method Now Utilized in Epigenetics Research and Characterization of Circulating Tumor Cells

  • Targeting Circulating Tumor Cells

    Circulating tumor cells (CTCs), released into the blood from primary and metastatic tumors, are rare but important. Scientists believe that CTCs may provide answers not only in understanding their role in establishing metastatic tumors, but also in revolutionizing cancer patient care. Caifu Chen, Ph.D., senior director of genomic assays R&D, Life Technologies, said, “Scientific communities and the biotech industry are racing to develop a novel device to accurately count and molecularly characterize the CTCs so that doctors can treat cancer patients early, individually, and, therefore, more effectively.”

    According to Dr. Chen, CTCs have unique signatures in mutation and gene-expression profiles. “We’re developing a new TaqMan® assay technology called RT-qCastPCR, which is capable of digital counting and cancer mutation detection of rare CTCs directly from blood.”

    This technology is based upon castPCR, a homogenous assay that combines competitive allele-specific TaqMan qPCR with an allele-specific minor groove binder as a blocker to suppress nonspecific amplification. “We have already validated castPCR assays for a hundred cancer mutations. Some assays can detect one to five mutant molecules in a million copies of wild-type background. CastPCR assays for 44 mutations of KRAS, EGFR, and BRAF genes were released commercially for early access in April.”

    Even though the new RT-qCastPCR technology is still at an early stage, the company believes that digital counting and mutation characterization of CTCs in blood could significantly improve CTC research. “This provides direct analysis of CTCs in blood, more sensitive and specific CTC counting based on multiple markers, mutation profiles of every detectable CTC, and a simple TaqMan workflow with answers in a few hours rather than days, as is currently on the market.”

  • mRNA Expression Profiling

    Sabine Lohmann and colleagues from Roche Pharma and Ludwig-Maximillian University discussed the use of customized, function-tested RealTime ready assays for gene-expression analysis in biomarker research and early drug development. They noted that the investigation of mRNA expression profiles in these two areas is of particular interest.

    According to the scientists, RealTime ready custom panels offer a variety of intron-spanning RT-qPCR assays covering the major signal transduction pathways, including those relevant for oncology research. The custom panels are composed of ready-to-use LightCycler® 480 multiwell plates containing preplated qPCR assays for human, mouse, and rat targets that have been selected by the user.

    The research team explained that they have established various workflows applicable for gene-expression analysis in biomarker research as well as in preclinical and early clinical studies. To illustrate, they said that preclinical drug development was started by profiling gene expression using a multiparameter panel (93 target and 3 housekeeping genes) covering the pathway of interest in a broad screening approach. The goal was to generate a first hypothesis for predictive and pharmacodynamic markers, as well as to provide a workflow with the highest convenience and throughput for this screening approach, they added.

    This first step was performed in vitro with tumor cell lines treated with the compound of interest. A workflow protocol was developed, starting with an automated RNA extraction on the MagNA Pure LC instrument in combination with pre-plated RealTime ready customized assays in a 384-well format. The relative gene expression already had been analyzed. After selecting a set of parameters, this first hypothesis was verified using an in vivo mouse model. Xenograft-derived fresh frozen tissue samples were used to test for selected biomarkers.

    The team next moved to early clinical development using the first set of clinical samples, formalin-fixed paraffin-embedded (FFPE) tissue derived from various tumor entities. They explained that tumor-derived FFPE tissue is the most relevant sample material for the development of therapeutic compounds in clinical trials or in biomarker research.

    RNA extraction from FFPE tissue (e.g., 10 µm sections) using the High Pure RNA Paraffin Kit was followed by qRT-PCR analysis. Then the researchers combined this workflow with function-tested RTR assays.

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