Detection and quantification of mRNA in single human polar bodies

A minimally invasive test of gene expression during oogenesis

Peter C. Klatsky, Gary M. Wessel, Sandra A. Carson

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Proteins and mRNA produced in oogenesis support embryonic development until the zygotic transition, 3 days after fertilization. Since polar bodies can be biopsied with little if any harm to the oocyte, we tested the hypothesis that mRNA originating from expression in the meiotic oocyte is present and detectable in a single polar body prior to insemination. Human oocytes were obtained from patients undergoing controlled ovarian hyperstimulation and intracytoplasmic sperm injection. Immature oocytes were cultured overnight and inspected the following day for maturation. Metaphase II oocytes underwent polar body biopsy followed by reverse transcription without RNA isolation. Sibling oocytes were similarly prepared. Complementary DNA from all samples were pre-amplified over 15 cycles for candidate genes using selective primers. Real-time PCR was performed to detect and quantify relative single-cell gene expression. Polar body mRNA was detected in 11 of 12 candidate genes. Transcripts that were present in greater abundance in the oocyte were more likely to be detected in qPCR replicates from single polar bodies. Pre-amplification of cDNA synthesized without RNA isolation can facilitate the quantitative detection of mRNA in single human polar bodies.

Original languageEnglish (US)
Article numbergaq077
Pages (from-to)938-943
Number of pages6
JournalMolecular Human Reproduction
Volume16
Issue number12
DOIs
StatePublished - Dec 2010
Externally publishedYes

Fingerprint

Polar Bodies
Oogenesis
Human Body
Oocytes
Gene Expression
Messenger RNA
Complementary DNA
RNA
Intracytoplasmic Sperm Injections
Insemination
Metaphase
Fertilization
Genes
Reverse Transcription
Embryonic Development
Siblings
Real-Time Polymerase Chain Reaction
Biopsy

Keywords

  • Gene expression
  • Infertility
  • Meiosis
  • Oocyte
  • Oocyte quality

ASJC Scopus subject areas

  • Molecular Biology
  • Embryology
  • Cell Biology
  • Genetics
  • Developmental Biology
  • Reproductive Medicine
  • Obstetrics and Gynecology

Cite this

Detection and quantification of mRNA in single human polar bodies : A minimally invasive test of gene expression during oogenesis. / Klatsky, Peter C.; Wessel, Gary M.; Carson, Sandra A.

In: Molecular Human Reproduction, Vol. 16, No. 12, gaq077, 12.2010, p. 938-943.

Research output: Contribution to journalArticle

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