Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling

S, Mao; Goodrich, Robert; Hauser, Russ; Schrader, Steven M.; Chen, Zhen; Krawetz, Stephen A.

doi:10.3109/19396368.2013.817626

Cited by 40 publications

(34 citation statements)

References 25 publications

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“…Despite this cache of sperm miRNA expression information, cross-species comparisons using these existing data would be, as discussed previously, intrinsically unreliable because of differences in methodology [20, 21, 23–27]. Using the standardized miRNA expression data available in SpermBase, we were able to identify 67 miRNAs that were present in the total sperm samples of all four species (Fig.…”

Section: Resultsmentioning

confidence: 97%

“…Compared to other cell types, sperm contain fewer RNAs, and some RNAs appear to be localized to the nucleus and associated protamine-packed chromatin highly enriched in disulfide bonds resistant to lysis by detergents [4, 18, 19]. Therefore, it is no surprise that different RNA extraction procedures have been shown to lead to highly variable sperm-borne RNA contents; this issue is compounded by the inherent heterogeneity of RNA populations among individual sperm samples [20–24]. Additionally, a sperm RNA isolation protocol that works for one species does not necessarily work for another, because of interspecies differences in sperm morphology and chromatin condensation [25–27].…”

Section: Introductionmentioning

confidence: 99%

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SpermBase: A Database for Sperm-Borne RNA Contents

Schuster

Tang

Xie

et al. 2016

Biology of Reproduction

109

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Since their discovery approximately three decades ago, sperm-borne RNAs, both large/small and coding/noncoding, have been reported in multiple organisms, and some have been implicated in spermatogenesis, early development, and epigenetic inheritance. Despite these advances, isolation, quantification, and annotation of sperm-borne RNAs remain nontrivial. The yields and subspecies of sperm-borne RNAs isolated from sperm can vary drastically depending on the methods used, and no cross-species analyses of sperm RNA contents have ever been conducted using a standardized sperm RNA isolation protocol. To address these issues, we developed a simple RNA isolation method that is applicable to sperm of various species, thus allowing for reliable interspecies comparisons. Based on RNASeq analyses, we established SpermBase (www.spermbase.org), a database dedicated to sperm-borne RNA profiling of multiple species. Currently, SpermBase contains large and small RNA expression data for mouse, rat, rabbit, and human total sperm and sperm heads. By analyzing large and small RNAs for conserved features, we found that many sperm-borne RNA species were conserved across all four species analyzed, and among the conserved small RNAs, sperm-borne tRNA-derived small noncoding RNAs and miRNAs can target a large number of genes known to be critical for early development.

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Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

SpermBase: A Database for Sperm-Borne RNA Contents

Schuster

Tang

Xie

et al. 2016

Biology of Reproduction

109

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“…RNA data for sperm, testes, and seminal fluid were generated and analyzed as described Mao et al 2013;Sendler et al 2013;Johnson et al 2015) and are available in the Gene Expression Omnibus repository (GSE62874: human sperm and seminal fluid; and GSE69434: human testes). Analysis of transcripts from testes and the different fractions of semen separated by ultracentrifugation was performed as described previously (Sendler et al 2013;Johnson et al 2015).…”

Section: Semen-omic Data and Analysesmentioning

confidence: 99%

The protein and transcript profiles of human semen

2015

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The increasing use of "-omics" (genomic, transcriptomic, proteomic, epigenomic, and metabolomic) high-throughput measurement technologies over the past decade is beginning to reveal the complexity of human biology and physiology through the interactions of DNA, RNA, related proteins and small molecules. In reproductive medicine, the majority of this work, has thus far focused on the female factors, e.g., the oocyte, since they provide both the environment and the majority of elements required for embryogenesis. State-of-the-art sequencing and computational analyses have enabled a deeper understanding of the underlying components. Contrary to being simply a silent delivery vehicle to the oocyte of the packaged male DNA, sperm provide both a specific epigenetically marked genome together with a complex population of RNAs and proteins that are crucial for early embryogenesis. In addition to the sperm, seminal fluid appears to serve multiple roles providing a supplementary series of components that allow the sperm to successfully reach and fertilize the oocyte and prepare the female immune system to tolerate the semiallosteric embryo. A global analysis and review of what is presently known regarding the unique role of each component of the male factor and their associated interactions begins to shed light on this emergent field.

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“…RNA-Seq has been applied to identify potential functions, transcript abundance and intactness, and particular characteristics of transcripts retained in spermatozoa [Jodar, et al 2013; Johnson et al 2011; Sendler, et al 2013]. Compared with typical somatic cells, sperm RNA has two features: (1) low quantity of RNA per cell (~50 fg) and (2) exist in a fragmented or partially degraded state [Johnson, et al 2011; Mao et al 2013; Sendler et al 2011] compared with RNA from typical somatic cells. Both characteristics make deep sequencing of sperm RNA more complicated, but also make sperm an ideal paradigm for the comparison of sequencing methodologies for low-input and fragmented samples.…”

Section: Introductionmentioning

confidence: 99%

A comparison of sperm RNA-seq methods

Sendler

Goodrich

et al. 2014

Systems Biology in Reproductive Medicine

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A significant challenge to the effective application of RNA-seq to the complete transcript analysis of low quantity and/or degraded samples is the amplification of minimal input RNA to enable sequencing library construction. Several strategies have been commercialized in order to facilitate this goal. However, each strategy has its own specific protocols and methodology, and each may introduce unique bias and in some cases show specific preference for a collection of sequences. Our wider investigation of human spermatozoal RNAs was able to reveal their complexity despite being generally characterized by low quantity and high fragmentation. In this study, four commercially available RNA-seq library amplification protocols for the preparation of low quantity/highly fragmented samples - SMARTer™ Ultra Low RNA (SU) for Illumina® Sequencing (Clontech Laboratories, Inc.), SeqPlex RNA Amplification (SP) (Sigma-Aldrich Co.), Ovation® RNA-Seq System V2 (OR), and Ovation® RNA-Seq FFPE System from NuGEN (FFPE) (NuGEN Technologies, Inc.) - were assessed using human sperm RNAs. Further investigation analyzed the effects on the end results of two different library preparation methods - Encore NGS Multiplex System I (Enc) and Ovation Ultralow Library Systems (UL) (NuGEN Technologies, Inc.) - that appeared best suited to this type of RNA, along with other potential confounding factors such as Formalin Fixed Paraffin Embedded (FFPE) preservation. Our results indicate that for each library preparation protocol, the differences in the initial amount of input RNA and choice of RNA purification step do not generate marked differences in terms of RNA profiling. However, substantial disparity is introduced by individual amplification methods prior to library construction. These significant differences may be caused by the different priming methods or amplification strategies used in each of the four different protocols examined. The observation of intrasample variation introduced by the choice of protocol highlights the role that external factors play in planning and reliable interpretation of results of any RNA-seq experiment.

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Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling

Cited by 40 publications

References 25 publications

SpermBase: A Database for Sperm-Borne RNA Contents

SpermBase: A Database for Sperm-Borne RNA Contents

The protein and transcript profiles of human semen

A comparison of sperm RNA-seq methods

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