Total RNA isolation from stallion sperm and testis biopsies

Das, Pranab Jyoti; Paria, Nandina; Gustafson-Seabury, Ashley; Vishnoi, Monika; Chaki, Sankar P.; Love, Charles C.; Varner, D.D.; Chowdhary, Bhanu P.; Raudsepp, Terje

doi:10.1016/j.theriogenology.2010.04.023

Cited by 70 publications

(50 citation statements)

References 23 publications

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“…Genomic DNA contamination of the samples was evaluated using real-time PCR (Light cycler 480; Roche) using a set of (Table 1) and spanning an intron; different PCR products were amplified from RNA (167 bp) and genomic DNA (351 bp), as described previously (Das et al 2010). Reactions for real-time PCR were performed in a final volume of 12 mL using 6 mL Kapa SYBR Fast (KAPA BIO-SYSTEMS), 0.6 mL each primer, 2.8 mL distilled water and 2 mL cDNA, which was replaced with distilled water as a negative control and with equine genomic DNA as a positive control.…”

Section: Quantitative Real-time Pcrmentioning

confidence: 99%

Implication of transcriptome profiling of spermatozoa for stallion fertility

Suliman

Becker

Wimmers

2018

Reprod. Fertil. Dev.

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Abstract. Poor fertility of breeding stallions is a recognised problem in the equine industry. The aim of the present study was to detect molecular pathways using two groups of stallions that differed in pregnancy rates as well as in the proportion of normal and motile spermatozoa. RNA was isolated from spermatozoa of each stallion and microarray data were analysed to obtain a list of genes for which transcript abundance differed between the groups (P #0.05, fold change $1.2). In all, there were 437 differentially expressed (DE) genes between the two groups (P # 0.05, fold change $1.2). Next, the DE genes were analysed using Database for Annotation, Visualisation, and Integrated Discovery (DAVID). Finally, ingenuity pathways analysis (IPA) was used to identify top biological functions and significant canonical pathways associated with the DE genes. Analysis using the DAVID database showed significant enrichment in the gene ontology (GO) term 'RNA binding' (P ¼ 0.05) and in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway cytokinecytokine receptor interaction (P ¼ 0.02). Furthermore, IPA analysis showed interconnected biological functions and canonical pathways involved in the regulation of spermatogenesis and male fertility. In addition, significantly enriched metabolic pathways were identified. In conclusion, the present study has identified, for the first time, molecular processes in stallion spermatozoa that could be associated with stallion fertility.

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Section: Quantitative Real-time Pcrmentioning

confidence: 99%

Implication of transcriptome profiling of spermatozoa for stallion fertility

Suliman

Becker

Wimmers

2018

Reprod. Fertil. Dev.

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“…Different researchers have their own opinion about origin of the traces of these spermatozoal transcripts in a transcriptionally quiescent sperm cell; Kramer and Krawetz (1997) proposed those as remnant untranslated RNAs during spermatogenesis, and are strongly appreciated after observing same spermatozoan transcript in both testes. Uncovering of spermatozoal RNA transcripts and their probable role in poultry and other bird species as already stated among other vertebrates (Goodrich et al, 2007;Gilbert et al, 2007;Lalancette et al, 2008;Yang et al, 2009;Das et al, 2010) may provide important breakthrough in identifying fertility biomarkers. In this review, we discuss the possibilities of using sperm RNAs as fertility/ infertility biomarkers based on the available literature for mammalian and other species because few reports are available on this particular subject on chicken sperm.…”

Section: Introductionmentioning

confidence: 94%

Sperm RNA: a New Class of Fertility Biomarkers for Birds

Shafeeque

2014

Adv. Anim. Vet. Sci.

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Leeuwenhoek discovered and named spermatozoa but could not convince the scientific world the potentiality of the sperm. Similarly, transcriptionally quiescent genetic material was marked by a state of tranquil among researchers until the breakthrough of RNA transcript residing inside the sperm. Even though, queries and enigmas highlight throughout sperm research, most reports favor us in thinking about role of these transcripts in fertilization and early embryonic development. Many sperm transcripts have been confirmed as bio-fertility marker in human and other mammalian specie. With this review, we are attempting to summarize spermatozoal work conducted so far in other vertebrate classes and thus representing a potential to serve as bio-fertility marker through such a non-invasive approach in birds. All copyrights reserved to Nexus® academic publishers

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“…Minogue et al [61] Rat NP, AF and AC tissues Modified TRIspin method Lee et al [128] In this study, we chose to use TRIzol for the RNA extraction as some studies have suggested the yield of RNA obtained using the guanidine isothiocyanate (GITC) based method can be higher than the column based method for some samples [129][130][131][132]. The studies in Table 3-2 either used TRIzol extraction or TRIspin method for RNA isolation.…”

Section: Tissue Extraction Referencementioning

confidence: 99%

“…RNeasy) are two commonly used methods for RNA extraction. Some studies have suggested the yield of RNA obtained using the GITC-based method can be higher than the column based method for some samples [129][130][131][132]. Thus we used GITC based TRIzol to lyse the cells.…”

Section: Comparison Of Rna From Propanol Precipitation and Column Basmentioning

confidence: 99%

“…In some situations, only a small number of cells is available for obtaining RNA such as cells sorted using fluorescent assisted cell sorting (FACS) [152], from tissues with a low cell density such as cartilage [153] and intervertebral disc [26] and when numerous cell culture conditions are tested with limited cell source. Some studies have suggested the yield of RNA obtained using the guanidine isothiocyanate (GITC) based method is higher than the column based method for some samples [129][130][131][132]. Besides, TRIzol (a GITC based reagent) allows the simultaneous extraction of RNA, DNA and protein from the same sample [154].…”

Section: Cryosection the Tissues And Lyse The Cells In Trizolmentioning

confidence: 99%

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Technique and method development for intervertebral disc (IVD) research

Lee¹,

李芷恩²

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Intervertebral disc (IVD) degeneration is one of the major causes of low back pain. The direct and indirect cost of managing back pain posts heavy socioeconomic burden to the society. Improved technologies and techniques together with well documented experiments will benefit the research field by saving effort in doing the optimization in every laboratory and avoiding experiment failure due to incomplete understanding of the procedures. These can accelerate scientific discovery, reduce the sacrifice of animals and enable a more effective use of funding.Nucleus pulposus (NP) is the central part of IVD. Differences in matrix compositions in human NP clinical samples demand different cell isolation protocols for optimal results but there is no clear guide about this to date. Sub-optimal protocols may result in low cell yield, limited reliability of results or even failure of experiments. We experimented different isolation protocols to study different parameters involved and suggested some rules for cell isolation in three main applications: RNA extraction for phenotyping, cell isolation for cell culture, and characterization by flow cytometry.In addition, instead of extracting RNA from isolated cells, extraction of RNA from tissues directly may avoid the change of RNA levels during the cell isolation process.However, extraction of RNA directly from human and large animal IVD tissue is technically challenging due to its tough nature, low cell-to-matrix ratio and high proteoglycan content. Thus we developed a method for RNA extraction from bovine disc tissues by integrating the use of cryosectioning, additional phase separation and high salt precipitation into conventional guanidinium thiocyanate based method. With this method, RNA could be extracted from the NP tissue directly but the concentration was low. A shift toward 270 nm was observed in its UV spectrum which was due to phenol contamination. This caused an overestimation of RNA concentration. Hence we developed a computational method based on UV spectra for correcting the In short, different methods related to IVD research were developed and optimized. With improved methods together with a better understanding of the underlying rationale, researchers can save time and cost in their experiments and reduce experiment failure rate. This will help to accelerate researches. New methods also enable studies which were not feasible in the past.(484 words)

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Total RNA isolation from stallion sperm and testis biopsies

Cited by 70 publications

References 23 publications

Implication of transcriptome profiling of spermatozoa for stallion fertility

Implication of transcriptome profiling of spermatozoa for stallion fertility

Sperm RNA: a New Class of Fertility Biomarkers for Birds

Technique and method development for intervertebral disc (IVD) research

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