title: Evaluation of ribonucleic acid amplification protocols for human oocyte transcriptome analysis.
abstract: OBJECTIVE
  To develop a reliable, reproducible, and sensitive method for investigating gene-expression profiles from individual human oocytes.
  
  
  METHODS
  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.
  
  
  METHODS
  University medical center and university microarray laboratory.
  
  
  METHODS
  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.
  
  
  METHODS
  None.
  
  
  METHODS
  Amplification efficiency and microarray profiles.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands; MicroArray Department and Integrative Bioinformatics Unit, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Eleni
authors.initials: E
authors.lastname: Mantikou
authors.affiliation: MicroArray Department and Integrative Bioinformatics Unit, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands; Netherlands Bioinformatics Center, Nijmegen, the Netherlands.
authors.forename: Oskar
authors.initials: O
authors.lastname: Bruning
authors.affiliation: Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: Mastenbroek
authors.affiliation: Center for Reproductive Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.
authors.forename: Sjoerd
authors.initials: S
authors.lastname: Repping
authors.affiliation: MicroArray Department and Integrative Bioinformatics Unit, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands; Netherlands Bioinformatics Center, Nijmegen, the Netherlands.
authors.forename: Timo Markus
authors.initials: TM
authors.lastname: Breit
authors.affiliation: MicroArray Department and Integrative Bioinformatics Unit, Swammerdam Institute for Life Sciences, Faculty of Science, University of Amsterdam, Amsterdam, the Netherlands. Electronic address: m.dejong@genomescan.nl.
authors.forename: Mark
authors.initials: M
authors.lastname: de Jong
mesh_terms: D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D009865:Oocytes / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D059467:Transcriptome
chemical_list: D005819:Genetic Markers; D012333:RNA, Messenger
keywords: Gene expression; mRNA amplification; microarray; single human oocytes; validation