Optimization of a microarray based approach for deriving representative gene expression profiles from human oocytes.
The purpose of the present study was to optimize a protocol for deriving reproducible and representative gene expression profiles from very rare research samples of human oocytes using microarrays. Immature oocytes produced as a result of administration of gonadotrophins for the treatment of infertility were donated to research. Linear amplification (L-amp) and exponential amplification (E-amp) were both capable of generating sufficient product for hybridization to the microarrays even from the low amount of template mRNA present in a single human oocyte. Slightly higher numbers of transcripts were detected by microarray following linear rather than E-amp but both techniques generated a product with reliably reproducible sensitivity and fidelity providing oocytes were pooled in minimum numbers of three. The majority of the variance associated with amplification and hybridization to arrays comes from the molecular processing. Slightly greater additional variance is associated with biological differences in immature oocytes from the same or different donors. The findings suggest that representative gene expression profiles can be generated from human oocytes for comparative purposes following L-amp and hybridization to microarray. However, oocytes must be pooled for the starting template for each array and sufficient independent microarray experiments performed to minimize the variance associated with molecular processing.
Jones
Gayle M
GM
Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Australia. gayle.jones@med.monash.edu.au
Song
Bi
B
Cram
David S
DS
Trounson
Alan O
AO
D005260:Female; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D014644:Genetic Variation; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009865:Oocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis
D012333:RNA, Messenger