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.

Developmental RNA-Seq transcriptomics of haploid germ cells and spermatozoa uncovers novel pathways associated with teleost spermiogenesis

“…1 e, inset). These data, together with immunofluorescence microscopy examination, indicate that these samples were almost devoid of non-sperm cells 36 .…”

The Effects of Storage in vitro on Functions, Transcriptome, Proteome, and Oxidation Resistance of Giant Grouper Sperm

“…Based on the computational formula of TPM, it is directly proportional to the number of transcripts. In addition, spermatozoa contained few or even no rRNA (Miller et al, 2005;Fang et al, 2014;El Fekih et al, 2017), which provides a favorable condition for the estimation of consistent transcripts. The average number of transcripts in each spermatozoon can be estimated based on TPM values and average RNA contents in each spermatozoon.…”

High‐quality human and rat spermatozoal RNA isolation for functional genomic studies

“…A plethora of data exists on sperm RNAs using different species and different targets such as mRNA, piwiRNA and miRNA, however they all face similar technical difficulties due to the unique biology of the sperm itself [25, 41]. To this, recent studies have tried to establish a standard, across species, protocol to extract sperm RNA [18, 22, 24, 42]. Consequently, the development of standardized sperm RNA preparation and isolation methods to deliver highly purified and intact RNA is of absolute importance.…”

“…Despite the absence of the ribosomal RNA in spermatozoa, spectral analysis of RNA abundance by size is still useful to assess sperm RNA quality and purity since the presence of the ribosomal subunits 18S and 28S may indicate somatic cell RNA contamination. A spermatozoon contains far less RNA than a somatic cell [11, 14, 42], and collection from rat epididymal fluid, as well as human ejaculate contain somatic cells along with spermatozoa; therefore, sperm purification in the RNA isolation protocol is an essential step. These observations are in conflict with a previous study that reported that ribosomal RNA 18S but not 28S is present in purified spermatozoa [19].…”