title: Sperm RNA preparation for transcriptomic analysis: Review of the techniques and personal experience.
abstract: In the last 10 years, several approaches, including microarrays, have been applied to investigate sperm transcript levels. However, success using microarray profiling is highly dependent of the quality of the RNA obtained. Therefore, the development of methods that deliver highly purified and intact RNA is of utmost importance. The three steps used to achieve this goal, purification of spermatozoa, RNA extraction and evaluation of RNA quality, are reviewed. Following that review and preliminary experiments, we processed sperm samples from seven normozoospermic men with a combination of gradient centrifugation and somatic cell lysis. RNA was extracted using the NucleoSpin RNA XS kit (Macherey-Nagel) and its purity checked using the BioAnalyzer. Hybridisation was done on Agilent SurePrint G3 Human GE 8 × 60K V2 microarrays. We identified 900 transcripts among the 1000 high abundance sperm transcripts reported in the literature. These genes are known to be involved in several biological processes, notably spermatogenesis, transcription regulation, cell growth and differentiation, sperm motility and capacitation, fertilisation, and embryogenesis. Therefore, our methodology is highly suitable for sperm transcriptomic analyses and can be used, notably, to compare mRNA profiles between fertile and infertile males.
authors.affiliation: Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, France.|Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France.|Laboratoire de Cytogénétique, Génétique Moléculaire et Biologie de la Reproduction Humaines, CHU Farhat Hached Sousse, Université de Monastir, Monastir, Tunisia.
authors.forename: S
authors.initials: S
authors.lastname: El Fekih
authors.affiliation: Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, France.|Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France.
authors.forename: M-H
authors.initials: MH
authors.lastname: Nguyen
authors.affiliation: Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, France.|Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France.|Service de Cytogénétique et Biologie de la Reproduction, Hôpital Morvan, CHRU Brest, Brest, France.
authors.forename: A
authors.initials: A
authors.lastname: Perrin
authors.affiliation: Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, France.|Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France.|Service de Cytogénétique et Biologie de la Reproduction, Hôpital Morvan, CHRU Brest, Brest, France.
authors.forename: D
authors.initials: D
authors.lastname: Beauvillard
authors.affiliation: Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, France.|Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France.|Service de Cytogénétique et Biologie de la Reproduction, Hôpital Morvan, CHRU Brest, Brest, France.
authors.forename: F
authors.initials: F
authors.lastname: Morel
authors.affiliation: Laboratoire de Cytogénétique, Génétique Moléculaire et Biologie de la Reproduction Humaines, CHU Farhat Hached Sousse, Université de Monastir, Monastir, Tunisia.
authors.forename: A
authors.initials: A
authors.lastname: Saad
authors.affiliation: Laboratoire de Cytogénétique, Génétique Moléculaire et Biologie de la Reproduction Humaines, CHU Farhat Hached Sousse, Université de Monastir, Monastir, Tunisia.
authors.forename: H
authors.initials: H
authors.lastname: Ben Ali
authors.affiliation: Faculté de Médecine et des Sciences de la Santé, Université de Brest, Brest, France.|Institut National de la Santé et de la Recherche Médicale (INSERM), U1078, Brest, France.|Service de Cytogénétique et Biologie de la Reproduction, Hôpital Morvan, CHRU Brest, Brest, France.
authors.forename: M
authors.initials: M
authors.lastname: De Braekeleer
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007248:Infertility, Male / Q000235:genetics / Q000378:metabolism*; D008297:Male; D012313:RNA / Q000032:analysis*; D013094:Spermatozoa / Q000378:metabolism*
chemical_list: D012313:RNA
keywords: RNA isolation; RNA quality; microarray; sperm RNA; sperm purification