Procedure for rapid oocyte selection based on quantitative analysis of cumulus cell gene expression.
OBJECTIVE
To develop a procedure for the analysis of gene expression in cumulus cells during the interval between ovum pick up and insemination to select the best oocytes for fertilization.


METHODS
Five RNA extraction methods, three reverse transcription procedures followed by Real-time quantitative PCR and one single-step mRNA quantification kit were tested to measure the expression of five genes in cumulus cells.


RESULTS
Two RNA extraction kits gave the best combination of efficiency and purity. One reverse transcription procedure gave the best speed and efficiency. The single-step kit required more biological material than would be available from single cumulus oocyte complexes (COCs).


CONCLUSIONS
Our test identified a combination of RNA extraction and reverse transcription procedures that enables the level measurement of 5 selected cumulus cell transcripts within 4 h. Using this combination it was possible to obtain a reliable quantification of gene expression in 44 out of 46 individual COCs collected from seven patients.
Ferrari
Stefania
S
Servizi Sforza srl, Medicina della Riproduzione, Milan, Italy. centrosterilita@policlinico.mi.it
Lattuada
Debora
D
Paffoni
Alessio
A
Brevini
Tiziana A L
TA
Scarduelli
Claudia
C
Bolis
Giorgio
G
Ragni
Guido
G
Gandolfi
Fulvio
F
D054885:Cumulus Cells / Q000378:metabolism*; D005260:Female; D005307:Fertilization in Vitro; D015870:Gene Expression; D006801:Humans; D054315:Oocyte Retrieval; D009865:Oocytes / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D012333:RNA, Messenger
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