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Comply with MIQE guidelines for qPCR: Agilent 2100 Bioanalyzer assessment of RNA integrity

By Ruediger Salowsky
Agilent Product Manager Bioanalyzer - RNA/DNA Solutions

Quantitative real-time polymerase chain reaction (qPCR) and microarray analysis have become essential for elucidating variations in gene expression. While guidelines that define the minimum information required for interpretation of microarray data have been available since 2001,[1] similar specifications for qPCR experiments have been developed only recently. In early 2009, a consortium of leading scientists who use qPCR, established specifications for the minimum information that you must report for a qPCR experiment that you wish to publish. These are the MIQE guidelines (for minimum information for publication of quantitative real-time PCR experiments). This article describes how the Agilent 2100 Bioanalyzer helps you meet these requirements.

Figure 1. Most major journals have adopted the MIQE guidelines, which were designed to improve the quality of publications about qPCR experiments.

In addition to a detailed description of potential sources of experimental variability, the MIQE guidelines contain a checklist that you should fill in and submit to the publisher when you send the manuscript. Among other items, nucleic acid extraction is an essential item on the checklist for qPCR assays. Especially for experiments that target mRNA profiles, assessment of RNA integrity is critical to ensure the relevance, accuracy, correct interpretation, and repeatability of a qPCR experiment.

Widely accepted method provides rapid results

The Agilent 2100 Bioanalyzer and its portfolio of Agilent RNA kits have become the industry-standard tool for RNA quality control, ensuring highest RNA sample integrity. Agilent developed the algorithm for RNA integrity number (RIN), which standardized the assessment of RNA quality. After you analyze an RNA sample on the 2100 Bioanalyzer, the software automatically assigns an integrity number from 1 to 10 (based on degradation level). Since its scientific publication in 2006, multiple journal articles have referred to the use of RIN and the implication of RNA degradation on differential gene expression experiments.[2,3,4]

The essential role the 2100 Bioanalyzer plays in complying with MIQE guidelines is discussed in a webinar by Professor Mikael Kubista from the TATAA Biocenter. In his presentation, Professor Kubista elucidates the relevance of sample management, the effect of RNA degradation on qPCR results, and how the TATAA Biocenter uses the Agilent 2100 Bioanalyzer for quality control of RNA samples.

Figure 2. This valuable webinar discusses causes of RNA degradation, and shows examples of the use of the 2100 Bioanalyzer and RIN to assess RNA integrity.

For researchers who work in qPCR, it is critical to ensure that RNA is of high quality. The 2100 Bioanalyzer, coupled with Agilent RNA kits and the use of RIN, form a complete analysis solution for rapid assessment of RNA quality for qPCR experiments. If you need to comply with MIQE guidelines, link to more information on use of the Agilent 2100 Bioanalyzer for RNA quality control, and on qPCR and RNA integrity checks.

References

1. A. Brazma, et al., “Minimum information about a microarray experiment (MIAME)—toward standards for microarray data,” Nature Genetics. 2001; 29:365-371.
2. S. Fleige and M. W. Pfaffl, “RNA integrity and the effect on the real-time qRT-PCR performance,” Mol Aspects Med. 2006 Apr-Jun;27(2-3):126-39.
3. C. Strand, J. Enell, I. Hedenfalk, and M. Fernö, “RNA quality in frozen breast cancer samples and the influence on gene expression analysis—a comparison of three evaluation methods using microcapillary electrophoresis traces,” BMC Mol Biol. 2007 May 22;8:38.
4. C. E. Jahn, A. O. Charkowski, and D. K. Willis, “Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation,” J Microbiol Methods. 2008 Oct;75(2):318-24.

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