Evaluation of ribonucleic acid amplification protocols for human oocyte transcriptome analysis
The authors validated protocols that produce reliable and reproducible gene-expression profiles of individual human oocytes.
Eleni Mantikou, Ph.D., Oskar Bruning, M.Sc., Sebastiaan Mastenbroek, Ph.D., Sjoerd Repping, Ph.D., Timo Markus Breit, Ph.D., Mark de Jong, Ph.D.
Volume 105, Issue 2, Pages 511-519
To develop a reliable, reproducible, and sensitive method for investigating gene-expression profiles from individual human oocytes.
Five commercially available protocols were investigated for their efficiency to amplify messenger RNA (mRNA) from 54 single human oocytes. Protocols resulting in sufficient yields were further validated using microarray technology. For the validation, mRNA was isolated from 25 human oocytes. To eliminate biological variation, RNA from 13 human oocytes was pooled together and split into 12 identical samples for further mRNA amplification. From 12 oocytes, mRNA was individually isolated.
University medical center and university microarray laboratory.
Couples undergoing intracytoplasmic sperm injection treatment were asked to donate their immature oocytes for research, and written informed consent was obtained in all cases. Seventy-nine human oocytes were used in total.
Main Outcome Measure(s):
Amplification efficiency and microarray profiles.
Two of the five protocols (WT-Ovation One-Direct and Arcturus RiboAMp HS Plus) resulted in sufficient yields and high success rates and were further validated for their performance in obtaining reliable, reproducible, and sensitive expression profiles from individual human oocytes. Evaluation of these two protocols demonstrated that they both displayed low technical variation and produced highly reproducible profiles (r ≥ 0.95). One of them identified significantly more transcripts but also had a higher number of false discoveries.
Two protocols generated ample amounts of mRNA for (quantitative) polymerase chain reaction, microarray, and sequencing techniques. Further validation using a design that discriminates between biological and technical variation showed that both protocols can be used for gene-expression profiling of individual human oocytes.