Reverse transcription priming methods affect normalisation choices for gene expression levels in oocytes and early embryos

Bo Yu, Helena T A van Tol, Tom A E Stout, Bernard A J Roelen

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Mammalian oocytes and embryos rely exclusively on maternal mRNAs to accomplish early developmental processes. Since oocytes and early embryos are transcriptionally silent after meiotic resumption, most of the synthesised maternal mRNA does not undergo immediate translation but is instead stored in the oocyte. Quantitative RT-PCR is commonly used to quantify mRNA levels, and correct quantification relies on reverse transcription and the choice of reference genes. Different methods for reverse transcription may affect gene expression determination in oocytes. In this study, we examined the suitability of either random or oligo(dT) primers for reverse transcription to be used for quantitative RT-PCR. We further looked for changes in poly(A) length of the maternal mRNAs during oocyte maturation. Our data indicate that depending on the method of reverse transcription, the optimal combination of reference genes for normalisation differed. Surprisingly, we observed a shortening of the poly(A) tail lengths of maternal mRNA as oocytes progressed from germinal vesicle to metaphase II. Overall, our findings suggest dynamic maternal regulation of mRNA structure and gene expression during oocyte maturation and early embryo development.

Original languageEnglish
Article numbergaab040
Pages (from-to)1-12
JournalMolecular Human Reproduction
Volume27
Issue number7
Early online date21 Jun 2021
DOIs
Publication statusPublished - 1 Jul 2021

Bibliographical note

Publisher Copyright:
© 2021 The Author(s). Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.

Keywords

  • bovine embryo
  • gene expression
  • maternal mRNA
  • poly(A) tail
  • qRT-PCR
  • reverse transcription

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