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 language | English (US) |
|---|---|
| Article number | gaq077 |
| Pages (from-to) | 938-943 |
| Number of pages | 6 |
| Journal | Molecular Human Reproduction |
| Volume | 16 |
| Issue number | 12 |
| DOIs | |
| State | Published - Dec 2010 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Detection and quantification of mRNA in single human polar bodies
T2 - A minimally invasive test of gene expression during oogenesis
AU - Klatsky, Peter C.
AU - Wessel, Gary M.
AU - Carson, Sandra A.
PY - 2010/12
Y1 - 2010/12
N2 - 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.
AB - 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.
KW - Gene expression
KW - Infertility
KW - Meiosis
KW - Oocyte
KW - Oocyte quality
UR - http://www.scopus.com/inward/record.url?scp=78649466893&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=78649466893&partnerID=8YFLogxK
U2 - 10.1093/molehr/gaq077
DO - 10.1093/molehr/gaq077
M3 - Article
C2 - 20837506
AN - SCOPUS:78649466893
VL - 16
SP - 938
EP - 943
JO - Molecular Human Reproduction
JF - Molecular Human Reproduction
SN - 1360-9947
IS - 12
M1 - gaq077
ER -