Comparison of spermatozoal RNA extraction methods in goats

Sahoo, Bijayalaxmi; Guttula, Praveen Kumar; Gupta, Mukesh Kumar

doi:10.1016/j.ab.2020.114059

Cited by 11 publications

(4 citation statements)

References 54 publications

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“…An alternative long-read sequencing technology, ONT sequencing, measures changes in ionic current as fragments move through protein nanopores, and does not depend on enzyme-based nucleotide incorporation or detection of fluorescence ( Ip et al, 2015 ). Due to its affordability and higher throughput – the ONT PromethION generates 20 times more reads per flow cell than the PacBio Sequel II ( Garalde et al, 2018 ) – ONT has been widely used for transcriptome annotation in organisms ranging from yeast to humans ( Sharon et al, 2013 ; Tilgner et al, 2014 ; Oikonomopoulos et al, 2016 ; Byrne et al, 2017 ; Jenjaroenpun et al, 2018 ; Kadobianskyi et al, 2019 ; Seki et al, 2019 ; Sessegolo et al, 2019 ; Workman et al, 2019 ; Müller et al, 2020 ; Sahoo et al, 2020 ), permitting the discovery of isoforms that were difficult to observe from short-read sequencing alone ( Steijger et al, 2013 ; Venturini et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

Large-Scale Multiplexing Permits Full-Length Transcriptome Annotation of 32 Bovine Tissues From a Single Nanopore Flow Cell

et al. 2021

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A comprehensive annotation of transcript isoforms in domesticated species is lacking. Especially considering that transcriptome complexity and splicing patterns are not well-conserved between species, this presents a substantial obstacle to genomic selection programs that seek to improve production, disease resistance, and reproduction. Recent advances in long-read sequencing technology have made it possible to directly extrapolate the structure of full-length transcripts without the need for transcript reconstruction. In this study, we demonstrate the power of long-read sequencing for transcriptome annotation by coupling Oxford Nanopore Technology (ONT) with large-scale multiplexing of 93 samples, comprising 32 tissues collected from adult male and female Hereford cattle. More than 30 million uniquely mapping full-length reads were obtained from a single ONT flow cell, and used to identify and characterize the expression dynamics of 99,044 transcript isoforms at 31,824 loci. Of these predicted transcripts, 21% exactly matched a reference transcript, and 61% were novel isoforms of reference genes, substantially increasing the ratio of transcript variants per gene, and suggesting that the complexity of the bovine transcriptome is comparable to that in humans. Over 7,000 transcript isoforms were extremely tissue-specific, and 61% of these were attributed to testis, which exhibited the most complex transcriptome of all interrogated tissues. Despite profiling over 30 tissues, transcription was only detected at about 60% of reference loci. Consequently, additional studies will be necessary to continue characterizing the bovine transcriptome in additional cell types, developmental stages, and physiological conditions. However, by here demonstrating the power of ONT sequencing coupled with large-scale multiplexing, the task of exhaustively annotating the bovine transcriptome – or any mammalian transcriptome – appears significantly more feasible.

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Section: Introductionmentioning

confidence: 99%

Large-Scale Multiplexing Permits Full-Length Transcriptome Annotation of 32 Bovine Tissues From a Single Nanopore Flow Cell

et al. 2021

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“…β-mercaptoethanol is deliberated a reducing agent that conclusively denatures RNases by disrupting the disulfide bonds and by removing the innate conformation required for enzyme action. Any RNases present in the sample to be extracted will be completely deactivated when combined with the strong denaturing properties of guanidiumisothiocyanate (GITC) and RLT buffer supplied in the RNeasy kits [47]. The addition of β-mercaptoethanol can act as a biological antioxidant by scavenging hydroxyl radicals and might be resulted in a high yield of RNA because of its property to break the disulfide bonds (S-S) and additional lysis and nuclear component dissociation.…”

Section: Discussionmentioning

confidence: 99%

Modified methods for bovine sperm RNA isolation for consistent quality and RNA yield

Dewry

Mohanty

Nath

et al. 2022

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Sperm mRNA transcriptional profiling can be used to evaluate the fertility of breeding bulls. This study aimed to compare the modified RNA isolation methods for higher RNA yield and quality from freshly ejaculated sperm of cattle and buffalo bull for further transcriptome analysis. Ten fresh ejaculates from each Sahiwal (n = 10 bulls x 10 ejaculates) and Murrah bulls (n = 10 bulls x 10 ejaculates) were used for RNA isolation. Swim-up technique was used for live sperm separation and recovery. From the recovered live sperm, total sperm RNA was isolated by conventional methods (TRIzol, Double TRIzol), membrane-based methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) and Kit (RNeasy mini) methods in fresh semen. Among different isolation methods; the membrane-based modified methods combined with TRIzol (RNeasy + TRIzol) with the addition of β-mercaptoethanol (BME) resulted significantly (P < 0.05) higher total RNA quantity (300-340 ng/µL) and better purity in different concentrations of spermatozoa viz., 30-40 million, 70-80 million and 300-400 million sperm. The study concluded that the inclusion of BME to the combined membrane-based methods with somatic cell lysis buffer solution was best for constant increased yield and purity of RNA isolation from Sahiwal cattle and Murrah buffalo bull sperm. This method will help with the interpretation of data from animal models and the consistency of clinical assessments of male factor fertility employing RNA molecular biomarkers.

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“…NGS has led to the characterization and quantification of many omics, including genomics (DNA sequencing), transcriptomics (RNA and cDNA sequencing), and epigenomics (ChiP-seq and DNA methylation analysis). More recently, third-generation sequencing methods involving single-molecule real-time (SMRT) sequencing have emerged (Sahoo et al, 2021a;Sahoo et al, 2021b). These newer SMRT sequencing methods do not require PCR amplification of templates and hence are devoid of PCR biases.…”

Section: Transcriptomicsmentioning

confidence: 99%

Applications of Omics Technology for Livestock Selection and Improvement

et al. 2022

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Conventional animal selection and breeding methods were based on the phenotypic performance of the animals. These methods have limitations, particularly for sex-limited traits and traits expressed later in the life cycle (e.g., carcass traits). Consequently, the genetic gain has been slow with high generation intervals. With the advent of high-throughput omics techniques and the availability of multi-omics technologies and sophisticated analytic packages, several promising tools and methods have been developed to estimate the actual genetic potential of the animals. It has now become possible to collect and access large and complex datasets comprising different genomics, transcriptomics, proteomics, metabolomics, and phonemics data as well as animal-level data (such as longevity, behavior, adaptation, etc.,), which provides new opportunities to better understand the mechanisms regulating animals’ actual performance. The cost of omics technology and expertise of several fields like biology, bioinformatics, statistics, and computational biology make these technology impediments to its use in some cases. The population size and accurate phenotypic data recordings are other significant constraints for appropriate selection and breeding strategies. Nevertheless, omics technologies can estimate more accurate breeding values (BVs) and increase the genetic gain by assisting the section of genetically superior, disease-free animals at an early stage of life for enhancing animal productivity and profitability. This manuscript provides an overview of various omics technologies and their limitations for animal genetic selection and breeding decisions.

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Comparison of spermatozoal RNA extraction methods in goats

Cited by 11 publications

References 54 publications

Large-Scale Multiplexing Permits Full-Length Transcriptome Annotation of 32 Bovine Tissues From a Single Nanopore Flow Cell

Large-Scale Multiplexing Permits Full-Length Transcriptome Annotation of 32 Bovine Tissues From a Single Nanopore Flow Cell

Modified methods for bovine sperm RNA isolation for consistent quality and RNA yield

Applications of Omics Technology for Livestock Selection and Improvement

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