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May 1, 2012 (Vol. 32, No. 9)

Drivers and Hurdles for qPCR

  • Standardization and Quality Control

    Although companies compete on achieving the highest reproducibility on their qPCR instruments, the qPCR measurement for molecular diagnostics is hardly ever the most confounding step of the testing protocol, provided an adequately performing instrument is used.

    Collection, transport, and storage of samples, nucleic acid extraction, and reverse transcription (in case of RNA) are steps that contribute much more confounding variation than qPCR. For example, blood collected in unstabilized matrices, such as EDTA tubes, shows seriously perturbed expression profiles due to the cells’ response to the foreign environment in the test tube. Serious preanalytical problems are also experienced in preserved tissue samples, where common fixation reagents such as formalin, selected primarily to preserve sample morphology, cause serious damage to the RNA.

    In Europe a major effort has been undertaken by the SPIDIA consortium to tackle the standardization and improvement of pre-analytical procedures for in vitro diagnostics. The program covers steps from the development of evidence-based guidelines to the identification of quality biomarkers and tools for the pre-analytical phase.

    Recently, SPIDIA presented an improved procedure for tissue preservation that maintains RNA at high quality, while preserving morphology. SPIDIA also coordinates proficiency ring trials in Europe helping routine laboratories evaluating their performance in the handling of and analyzing biological specimens with qPCR.

    The European Committee for Standardization (CEN) is a SPIDIA member and the consortium interacts closely with the Office of Biorepositories and Biospecimen Research (OBBR) of the National Cancer Institute and the Clinical Laboratory Standards Institute (CLSI).

    CLSI has released several guidelines relevant for molecular testing, many of which has been adapted by the FDA. These initiatives are most important to establish qPCR as a robust and reliable platform for routine diagnostics.

    For research applications the “MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments” have been developed to encourage better experimental practice and allow for more reliable and unequivocal interpretation of qPCR results.

    Several leading journals enforce MIQE and many more endorse them. Guidelines are good but not sufficient; we also need standards. Recently, the U.S. National Institute of Standards and Technology released the first viral DNA Standard Reference Material—for cytomegalovirus (CMV). The CMV was quantified as genome copies/volume using digital PCR (dPCR).

    In dPCR a sample is aliquoted into a large number of reaction chambers such that only some contain targeted DNA and give rise to product upon PCR amplification. Counting the number of positive reactions essentially corresponds to counting single DNA molecules, resulting in absolute quantification and traceability to the SI unit, the mole.

    qPCR technology is developing rapidly, although most current efforts are on upstream (pre-analytic) and downstream (data mining) processes, rather than on the qPCR technique itself which, I believe, has almost reached perfection.

    A lot of work remains to be done until the entire workflow is standardized. Even more challenging will be to develop standards for various important DNA and RNA targets. The guidelines under development now are an important step in the right direction.

  • Digital PCR Products Continue to Emerge

    Click Image To Enlarge +
    RainDance Technologies’ RainDrop generates up to 10 million picoliter-sized droplets per lane. Each droplet encapsulates a single molecule.

    Joining the firms that have been launching digital PCR technology is RainDance Technologies. The company recently introduced its new RainDrop™ digital PCR system, which enables digital answers across a number of applications including low-frequency tumor allele detection, gene expression, copy number variation, and SNP measurement.

    The RainDrop system, built on RainStorm™ picodroplet technology, generates up to 10 million picoliter-sized droplets per lane. Since each droplet encapsulates a single molecule, researchers can determine the absolute number of droplets containing specific target DNA and compare that to the number of droplets with background wild-type DNA. The instrument employs a two-color-per-marker approach and varying probe intensity method that is capable of multiplexing up to 10 markers.

    In a recent Lab on a Chip paper, scientists from Université de Strasbourg and Université Paris Descartes used the RainDance digital PCR technology to detect a single mutated copy of KRAS in a background of 200,000 wild-type copies. “With RainDance digital PCR, we were able to achieve absolute quantification of mutated and tumor-circulating DNA and improve the detection of a circulating tumor by comparing its proportion to nontumor DNA,” says Pierre Laurent-Puig, M.D., Ph.D., of the Université Paris Descartes. “Absolute quantification is critical, especially in research that lays the groundwork for future clinical applications, because it allows you to generate meaningful thresholds that will be required for prognostic and diagnostic tools.”

    Last fall, Life Technologies launched its digital PCR instrument, the Applied Biosystems QuantStudio™ 12K Flex Real-Time PCR system. This genetic analysis instrument can generate more than 12,000 high-quality TaqMan® data points per run, or up to 110,000 data points in an eight-hour workday, the firm reports. Users can also run digital PCR experiments on the instrument using nanofluidic consumables and dedicated analysis software for increased sensitivity and specificity.

    The nanoliter volumes required to conduct experiments on the QuantStudio 12K Flex OpenArray® plates make the instrument cost efficient, saving both reagent and samples, Life Tech notes. The system can accommodate any one of five different interchangeable blocks (OpenArray plates, TaqMan Array Card, 384-, 96 fast-, and 96-well plates) to match the size and type of experiment.

  • Click Image To Enlarge +
    Fluidigm uses its integrated fluidic circuits to perform digital PCR on up to 48 unique samples per chip, with each sample partitioned into 770 individual reaction chambers.

    Fluidigm is also a player in the digital qPCR arena. The firm uses its integrated fluidic circuits (digital array chips) to perform digital PCR on up to 48 unique samples per chip, with each sample being partitioned into 770 individual reaction chambers. Fluidigm has experimental chip formats that can change the number of samples per chip, as well as the number of reaction chambers per sample. With the company’s approach, a positive signal indicates the presence of a target, while the absence of signal indicates the lack of a target.

    Fluidigm’s digital PCR system is closed, minimizing the possibility of PCR-induced contamination. The firm says the workflow is simple; a single hands-on step is required per sample.

    Bio-Rad Laboratories offers the QX100™ Droplet Digital PCR™ (ddPCR) system. The company says that this system provides an absolute measure of target DNA molecules. Applications include enriching for rare target sequences, detecting small fold (1.2x) target differences, and determining copy number without a standard curve.

    Bio-Rad reports that the instrument’s sensitivity and precision arise from ddPCR’s ability to partition a sample into 20,000 droplets. Droplet Digital PCR allows researchers to detect 0.001% mutation fractions, completely resolve copy number variations, distinguish up to 10% differences in gene copy, and accurately quantify genes that differ by a single nucleotide, according to the company.

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