Detection and quantification of mRNA in single human polar bodies: a minimally invasive test of gene expression during oogenesis.
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.
Klatsky
Peter C
PC
Division of Reproductive Endocrinology and Infertility, Women and Infants Hospital, Alpert School of Medicine, Brown University, 101 Dudley Street, Providence, RI 02905, USA. pklatsky@wihri.org
Wessel
Gary M
GM
Carson
Sandra A
SA
D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D009866:Oogenesis / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*
D012333:RNA, Messenger