Multiplex degenerate PCR coupled with an oligo sorbent array for human endogenous retrovirus expression profiling.
Human endogenous retroviruses (HERVs) can be divided into distinct families of tens to thousands of paralogous loci. The expression of HERV elements has been detected in all tissues tested to date, particularly germ cells, embryonic tissues and neoplastic tissues. Hence, the study of HERV expression could represent added value in cancer diagnosis. We developed a quantitative assay combining a multiplex degenerate PCR (MD-PCR) amplification, based on the relative conservation of the pol genes, and a colorimetric Oligo Sorbent Array (OLISA). Nine HERV families were selected and amplification primers and capture probes were designed for each family. The features required to achieve efficient amplification of most of the elements of each HERV family and balanced co-amplification of all HERV families were analyzed. We found that MD-PCR reliability, i.e. equivalence of amplification and dose-effect relationship, relied on the adjustment of three critical parameters: the primer degeneracy, the relative concentration of each primer and the total amount of primers in the amplification mixture. The analysis of tumoral versus normal tissues suggests that this assay could prove useful in tumor phenotyping.
Pichon
Jean-Philippe
JP
Unité Mixte de Recherche 2714, CNRS-bioMérieux, IFR128 BioSciences Lyon-Gerland, ENS-Lyon 46 allée d'Italie, 69364 Lyon cedex 07, France.
Bonnaud
Bertrand
B
Cleuziat
Philippe
P
Mallet
François
F
D002454:Cell Differentiation; D045744:Cell Line, Tumor; D003124:Colorimetry; D017931:DNA Primers / Q000737:chemistry; D020077:Endogenous Retroviruses / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis* / Q000378:metabolism
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012334:RNA, Neoplasm