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Methods of RNA preparation affect mRNA abundance quantification of reference genes in pig maturing oocytes
Author(s) -
Wang YK,
Li X,
Song ZQ,
Yang CX
Publication year - 2017
Publication title -
reproduction in domestic animals
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.546
H-Index - 66
eISSN - 1439-0531
pISSN - 0936-6768
DOI - 10.1111/rda.12972
Subject(s) - reference genes , rna , gene expression , biology , gene , rna extraction , microbiology and biotechnology , messenger rna , lysis , glyceraldehyde 3 phosphate dehydrogenase , real time polymerase chain reaction , reverse transcription polymerase chain reaction , genetics
Contents To ensure accurate normalization and quantification of target RNA transcripts using reverse transcription quantitative polymerase chain reaction ( RT ‐ qPCR ), most studies focus on the identification of stably expressed gene(s) as internal reference. However, RNA preparation methods could also be an important factor, especially for test samples of limited quantity (e.g. oocytes). In this study, we aimed to select appropriate reference gene(s), and evaluate the effect of RNA preparation methods on gene expression quantification in porcine oocytes and cumulus cells during in vitro maturation. Expression profiles of seven genes ( GAPDH , 18S , YWHAG , BACT , RPL 4 , HPRT 1 and PPIA ) were examined, on RNA samples extracted from cumulus cells ( RN easy Kit) and oocytes ( RN easy Kit and Lysis Kit) during in vitro maturation, respectively. Interestingly, different RNA preparation methods were found to potentially affect the quantification of reference gene expression in pig oocytes cultured in vitro. After geNorm analyses, the most suitable genes for normalization were identified, GAPDH / 18S for cumulus cells and YWHAG / BACT for oocytes, respectively. Thus, our results provide useful data and information on the selection of better reference genes and RNA preparation method for related functional studies.