Sperm RNA preparation for transcriptomic analysis: Review of the techniques and personal experience

Summary 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 an...

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Bibliographic Details
Published in:Andrologia 2017-12, Vol.49 (10), p.e12767-n/a
Main Authors: El Fekih, S., Nguyen, M.‐H., Perrin, A., Beauvillard, D., Morel, F., Saad, A., Ben Ali, H., De Braekeleer, M.
Format: Article
Language:English
Subjects:
Animals
Capacitation
Centrifugation
DNA microarrays
Embryogenesis
Fertilization
Gene Expression Profiling - methods
Gene regulation
Humans
Infertility, Male - genetics
Infertility, Male - metabolism
Lysis
Male
microarray
Purification
Reviews
RNA - analysis
RNA isolation
RNA quality
Sperm
sperm purification
sperm RNA
Spermatogenesis
Spermatozoa - metabolism
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Summary:Summary 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.
ISSN:0303-4569
1439-0272
DOI:10.1111/and.12767