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Jens Björkman1, David Švec1,2, Robert Sjöback1, Emelie Lott1, Mikael Kubista1,2 1TATAA Biocenter AB, Sweden; 2Laboratory of Gene Expression, Institute of Biotechnology, Academy of Sciences of the Czech Republic, Prague, Czech Republic |
Abstract
Since the publication of the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines in 2009 users’ awareness and clients’ requests for quality control (QC) in qPCR has dramatically increased. To comply with MIQE professional providers of qPCR services and contract research organizations emphasize the different aspects of quality control in their offerings. TATAA Biocenter co-authored the MIQE- guidelines, was partner in the SPIDIA consortium (www.spidia.org), member of the workgroup drafting the forthcoming ISO guidelines for the pre-analytical steps in molecular diagnostics, and is certified according to ISO 17025. TATAA early identified the need for and importance of stringent quality control in qPCR and has focused on developing methods and tools to asses sample, assay and performance quality parameters.
In this work we have identified biomarkers resistant to nucleolytic RNA degradation that can be used to assess RNA quality in samples exposed to temperatures and conditions at which nucleases are active or freeze-thawing. We also developed protocol to assess physical and chemical degradation, such as damaged caused during tissue fixation. These methods will be presented demonstrating their advantages in terms of sensitivity, performance and ease of use compared to traditional methods such as capillary electrophoresis and the 5’-3’ assay.
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