PURPOSE: 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.
|Titolo:||Procedure for rapid oocyte selection based on quantitative analysis of cumulus cell gene expression|
|Settore Scientifico Disciplinare:||Settore VET/01 - Anatomia degli Animali Domestici|
|Data di pubblicazione:||lug-2010|
|Digital Object Identifier (DOI):||10.1007/s10815-010-9428-1|
|Appare nelle tipologie:||01 - Articolo su periodico|