Digital multiplexed mRNA analysis of functionally important genes in single human oocytes and correlation of changes in transcript levels with oocyte protein expression.
OBJECTIVE
To investigate functionally important transcripts in single human oocytes with the use of NanoString technology and determine whether observed differences are biologically meaningful.


METHODS
Analysis of human oocytes with the use of NanoString and immunoblotting.


METHODS
University-affiliated reproductive medicine unit.


METHODS
Women undergoing in vitro fertilization.


METHODS
Human oocytes were analyzed with the use of NanoString or immunoblotting.


METHODS
The abundance of transcripts for ten functionally important genes-AURKA, AURKC, BUB1, BUB1B (encoding BubR1), CDK1, CHEK1, FYN, MOS, MAP2K1, and WEE2-and six functionally dispensable genes were analyzed with the use of NanoString. BubR1 protein levels in oocytes from younger and older women were compared with the use of immunoblotting.


RESULTS
All ten functional genes but none of the six dispensable genes were detectable with the use of NanoString in single oocytes. There was 3- to 5-fold variation in BUB1, BUB1B, and CDK1 transcript abundance among individual oocytes from a single patient. Transcripts for these three genes-all players within the spindle assembly checkpoint surveillance mechanism for preventing aneuploidy-were reduced in the same oocyte from an older patient. Mean BUB1B transcripts were reduced by 1.5-fold with aging and associated with marked reductions in BubR1 protein levels.


CONCLUSIONS
The abundance of functionally important transcripts exhibit marked oocyte-to-oocyte heterogeneity to a degree that is sufficient to affect protein expression. Observed variations in transcript abundance are therefore likely to be biologically meaningful, especially if multiple genes within the same pathway are simultaneously affected.
Reproductive Medicine Unit, Elizabeth Garrett Anderson Wing, University College London Hospitals, London, United Kingdom.
Solon
S
Riris
NanoString Technologies, Seattle, Washington.
Philippa
P
Webster
Reproductive Medicine Unit, Elizabeth Garrett Anderson Wing, University College London Hospitals, London, United Kingdom; Institute for Women's Health, University College London, London, United Kingdom; School of Women's & Children's Health, University of New South Wales, Randwick, New South Wales, Australia. Electronic address: h.homer@unsw.edu.au.
Hayden
H
Homer
D000328:Adult; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D036103:Nanotechnology / Q000379:methods*; D009865:Oocytes / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*; D014158:Transcription, Genetic / Q000502:physiology*
D012333:RNA, Messenger
NanoString; expression profiling; gene expression; human oocytes; oocyte quality