Matthias W Sieber1,2, Peter Recknagel2, Florian Glaser2, Otto W. Witte1, Michael Bauer2, Ralf A. Claus2, Christiane Frahm1
1Hans Berger Clinic for Neurology, Jena University Hospital, Germany; 2Anaesthesiology and Intensive Care Medicine, Jena University Hospital, Germany
Reverse transcription followed by quantitative PCR (rt-qPCR) has become the state of the art tool forquantification of nucleic acids. However, there are still significant problems associated with its sensitivity, reproducibility, efficiency and the choice of an appropriate rt-qPCR kit. The purpose of this study is to give insights into strategies to optimize and validate the performance of currently available kits for rt-qPCR and to provide up-to-date information about the benefits, potentials and pitfalls of rt-qPCR assays. A selection of 9 cDNA synthesis and 12 qPCR kits were tested using samples obtained from three species (mouse, rat and human) and three transcripts (Gapdh, Actb and Hmbs) under highly standardized conditions. Kits with an outstanding performance were further analysed to identify the dynamic range for a reliable quantification of mRNA. Reverse transcription efficiency varied up to 90 fold depending on the choice of reverse transcriptase, priming strategy and assay volume. The qPCR kit test revealed variations in mean relative amplification efficiency ranging from 54% to 171%. We conclude that currently available kits for rt-qPCR vary considerably. However, with an appropriate validation strategy and knowledge about capabilities of a particular kit, sensitivity, efficiency and reliability could be significantly improved.
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