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September 15, 2011 (Vol. 31, No. 16)

Assuring Reliability of qPCR & RT-PCR Results

Use of Spectrophotometry on Nucleic Acid Samples Before Experiment Improves Outcome

  • The polymerase chain reaction (PCR) is a valuable tool used in both research and molecular diagnostic laboratories because of its specificity, efficiency, fidelity, and relative ease of use.

    Quantitative real-time PCR (qPCR) enables sensitive and accurate quantitative measurement of nucleic acids. Both qPCR and reverse transcriptase PCR (RT-qPCR) are used across a wide range of applications such as gene expression, SNP genotyping, copy-number analysis, pathogen detection, drug target validation, and measurement of RNA interference (RNAi).

    The quality of qPCR and RT-qPCR results can be negatively affected by many experimental variables. To ensure the validity of assay results, sample extraction and preparation steps must be closely monitored, and the starting material must be well characterized before performing RT and qPCR assays.

    Slight differences in pipetting, lack of instrument calibration, improper choice of reference genes, incorrect quantification, and/or use of impure nucleic acid templates can generate erroneous, but believable, results. Therefore, the use of standardized best practices to ensure reliable and meaningful results is recommended. To address the need for standardized qPCR practices, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines have been developed.

  • The MIQE Guidelines

    The MIQE guidelines define the minimum information necessary for proper evaluation of qPCR experiments and publications. By detailing all relevant experimental conditions and assay characteristics, the validity of the protocols used can be thoroughly assessed. Complete disclosure of all reagents, sequences, and analysis methods used enables other investigators to reproduce results.

    The guidelines address several general categories including sample preparation, QC of nucleic acids, RT, qPCR assay, and data analysis. They also include an extensive checklist of “essential” or “desirable” items to be included in each of the following sections: experimental design, sample, nucleic acid extraction, RT, target information, primers and probe, qPCR protocol, qPCR validation and data analysis.

    Most of the MIQE guidelines can be adopted quickly. Adoption of the “essential” items ensures that key variables affecting data quality are addressed, which, in turn, increases the degree of confidence in the results and the conclusions drawn. Adoption of some or all of the “desirable” items further ensures that a comprehensive approach is being taken to obtain quality data.

    For instance, the guidelines recommend the disclosure of the probe sequence as highly desirable and strongly encouraged, but not all vendors of predesigned assays provide this information. Thermo Fisher Scientific provides probe sequences for all Solaris qPCR gene-expression assays.

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