A comparison of sperm RNA-seq methods

Abstract

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.

SpermBase: A Database for Sperm-Borne RNA Contents

“…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.…”

“…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].…”

RNA sequencing as a powerful tool in searching for genes influencing health and performance traits of horses

“…To date, the RNA-seq method has been successfully used to investigate the transcriptome in almost all species around the world, starting with humans (Cheng et al 2014;Mao et al 2014), mice (Hoang et al 2014), pigs (Ropka-Molik et al 2014) cattle (McLoughlin et al 2014, and dogs (Davis and Ostrander 2014).…”

De Novo RNA Sequencing and Transcriptome Analysis of Monascus purpureus and Analysis of Key Genes Involved in Monacolin K Biosynthesis

“…purpureus by RNA-seq [29–31], which was then annotated using publicly available databases and tools to identify genes involved in the biosynthesis of fungal polyketone compounds that may be related to the tricarboxylic acid cycle [32]. To this end, mRNA was isolated from liquid medium during vegetative growth of M .…”