2017
DOI: 10.4314/sajas.v47i1.9
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Isolation of total ribonucleic acid from fresh and frozen-thawed boar semen and its relevance in transcriptome studies

Abstract: The main objective of this study was to isolate high-quality total ribonucleic acid (RNA) from raw fresh semen and frozen-thawed boar semen, using a protocol comprising the conventional TRIzol assay and a membrane-based technique, the PureLink RNA mini kit. Bioanalyzer profile revealed that the sperm RNA size distributions comprised mainly intact RNA ranging from 1500 to 1800 bp, without any detectable residual genomic deoxyribonucleic acid (DNA) or 28S ribosomal RNA (rRNA). Spectrophotometric quantifications … Show more

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Cited by 3 publications
(6 citation statements)
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“…The present study revealed the transcripts for 13,814 genes in crossbred bull sperm, which is similar to the earlier studies by and Selvaraju et al (2017) and Raval et al (2019) , but Card et al (2013) reported only 6,166 transcripts in bovine sperm. These variations might be due to season of sample collection ( Godia et al, 2019 ), state of spermatozoa (fresh or frozen) ( Chen et al, 2015 ; Card et al, 2017 ; Singh et al, 2019 ), method of RNA isolation ( Parthipan et al, 2015 ; Fraser et al, 2017 ), integrity of sperm RNA, RNA sequencing instrument ( Selvaraju et al, 2017 ), and library preparation methods ( Mao et al, 2014 ). A high proportion of protein coding ribosomal RNAs was detected as unique and upregulated in low-fertile sperm than high-fertile sperm.…”
Section: Discussionmentioning
confidence: 99%
“…The present study revealed the transcripts for 13,814 genes in crossbred bull sperm, which is similar to the earlier studies by and Selvaraju et al (2017) and Raval et al (2019) , but Card et al (2013) reported only 6,166 transcripts in bovine sperm. These variations might be due to season of sample collection ( Godia et al, 2019 ), state of spermatozoa (fresh or frozen) ( Chen et al, 2015 ; Card et al, 2017 ; Singh et al, 2019 ), method of RNA isolation ( Parthipan et al, 2015 ; Fraser et al, 2017 ), integrity of sperm RNA, RNA sequencing instrument ( Selvaraju et al, 2017 ), and library preparation methods ( Mao et al, 2014 ). A high proportion of protein coding ribosomal RNAs was detected as unique and upregulated in low-fertile sperm than high-fertile sperm.…”
Section: Discussionmentioning
confidence: 99%
“…In this study a column-based RNA purification protocol was used to isolate total RNA from the fresh spermatozoa of the PLW boars, according to a previously described study (Fraser et al 2017), with some modifications. The modified protocol was based on the application of the TRIzol/Pure Link RNA Mini kit (Invitrogen, Thermo Fisher Scientific Inc.) followed by a 2× DNase digestion procedure to ensure effective removal of genomic (g) DNA (Turbo DNase, Ambion, USA).…”
Section: Methodsmentioning
confidence: 99%
“…It has been suggested that the analysis of the sperm-derived RNAs might provide potential links between the sperm proteome and semen freezability [17,18,20,[46][47][48]. It should be underlined that the isolation of high-quality RNAs from fresh or frozen-thawed semen is important to assess the sperm gene expression [19,46,49,50]. However, to optimize the isolation protocol of total RNA from spermatozoa of diferent animal species, the procedure has to be modiied, and should incorporate a quality control reverse transcription polymerase chain reaction (RT-PCR) to avoid somatic cell contamination [17,19,46,49].…”
Section: Transcriptome Studies On Sperm-derived Rnasmentioning
confidence: 99%
“…It should be underlined that the isolation of high-quality RNAs from fresh or frozen-thawed semen is important to assess the sperm gene expression [19,46,49,50]. However, to optimize the isolation protocol of total RNA from spermatozoa of diferent animal species, the procedure has to be modiied, and should incorporate a quality control reverse transcription polymerase chain reaction (RT-PCR) to avoid somatic cell contamination [17,19,46,49]. Presently, human sperm transcripts are the best characterized among all mammals with respect to RNA sequence proiling.…”
Section: Transcriptome Studies On Sperm-derived Rnasmentioning
confidence: 99%
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