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Quantitative multiplex degenerate PCR for human endogenous retrovirus expression profiling

Nature Protocols volume 1, pages 2831–2838 (2006)Cite this article

Abstract

Expression of human endogenous retroviruses (HERV) has been recurrently observed during cellular differentiation or transformation processes in both cell culture and in vivo. Quantitative approaches that analyze variations in HERV transcription could therefore be valuable for cancer diagnosis. We have developed a quantitative assay combining multiplex degenerate PCR (MD-PCR) and a colorimetric Oligo Sorbent Array (OLISA). Quantification of the expression of these multifamily genes relies on the optimization of the amplification primer mix, that is, the primer degeneracy, the relative concentration of each primer and the total amount of primer. Amplification products of each of the nine studied HERV families are independently and specifically detected and quantified using the OLISA microarray. This method constitutes an improvement over previous pan-retrovirus amplification-based methods, which are mainly qualitative. Furthermore, as MD-PCR/OLISA simultaneously monitors several HERV families, it challenges single-family quantitative RT-PCR. Last, the protocol below provides general rules for the design of MD-PCR applications. Once primers have been designed and optimized, the procedure can be completed in 2 days.

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Figure 1: General outline of the MD-PCR/OLISA procedure.

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Acknowledgements

J.-Ph. Pichon was supported by doctoral fellowships from bioMérieux and l'Association Nationale de la Recherche Technique and from l'Association pour la Recherche sur le Cancer.

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  1. Unité Mixte de Recherche 2714 CNRS––bioMérieux, IFR128 BioSciences Lyon-Gerland, ENS-Lyon, 46 allée d'Italie, Lyon, 69364, Cedex 07, France

    Jean-Philippe Pichon, Bertrand Bonnaud & François Mallet

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  1. Jean-Philippe Pichon
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  2. Bertrand Bonnaud
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Correspondence to François Mallet.

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Pichon, JP., Bonnaud, B. & Mallet, F. Quantitative multiplex degenerate PCR for human endogenous retrovirus expression profiling. Nat Protoc 1, 2831–2838 (2006). https://doi.org/10.1038/nprot.2006.475

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