scholarly journals A molecular analysis of the population of mRNA in bovine spermatozoa

Reproduction ◽  
2007 ◽  
Vol 133 (6) ◽  
pp. 1073-1086 ◽  
Author(s):  
Isabelle Gilbert ◽  
Nathalie Bissonnette ◽  
Guylain Boissonneault ◽  
Maud Vallée ◽  
Claude Robert
Keyword(s):  
Complex Mixture ◽  
Pcr Amplification ◽  
Male Gamete ◽  
Molecular Size ◽  
Messenger Rnas ◽  
Rna Integrity ◽  
Rna Profiling ◽  
Cell Functions ◽  
Bovine Spermatozoa ◽  
Global Amplification

Spermiogenesis represents the transition from haploid spermatids to spermatozoa. This process entails an extreme condensation of the nucleus and a loss of nearly all cytoplasmic content. The presence of messenger RNAs in the spermatozoa has previously been shown. Generally, these transcripts are considered to be remnants of spermiogenesis. However, it has recently been proposed that there may exist a function for these sperm-associated RNAs. To address the possibility of a functional role for these transcripts, we sought to investigate and characterize the RNA pool found in bovine spermatozoa. The main goals of this study were to examine RNA integrity and survey the mRNA found in spermatids and spermatozoa. Assessment of mRNAs integrity was performed by three approaches: microelectrophoresis, comparative smearing after global amplification, and PCR amplification of target sequences located either in the 5′ or the 3′ ends, while mRNAs survey was performed by microarray hybridizations. RNA integrity studies in the spermatozoa showed a majority of low molecular size fragments indicating a natural segmentation of the mRNA population. The mRNA survey indicated that the sperm transcriptome harbors a complex mixture of messengers implicated in a wide array of cell functions and representing a large subset of transcripts found in spermatids. Subsequently, such sperm RNA profiling could allow the molecular diagnosis of male gamete quality.

scholarly journals A Universal Test to Determine the Integrity of RNA, and its Suitability for Reverse Transcription, in Animal Tissue Laboratory Specimens

2007 ◽  
Vol 19 (5) ◽  
pp. 459-464
Author(s):  
H. Jane Oakey
Keyword(s):  
Quality Assurance ◽  
16S Rrna ◽  
Reverse Transcription ◽  
False Negative ◽  
Pcr Amplification ◽  
Viral Rna ◽  
Test Results ◽  
Tissue Samples ◽  
Rna Integrity ◽  
Template Material

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

scholarly journals Expression and putative function of fibronectin and its receptor (integrin α5β1) in male and female gametes during bovine fertilization in vitro

Reproduction ◽  
2009 ◽  
Vol 138 (3) ◽  
pp. 471-482 ◽  
Author(s):  
Mirjan Thys ◽  
Hans Nauwynck ◽  
Dominiek Maes ◽  
Maarten Hoogewijs ◽  
Dries Vercauteren ◽  
...  
Keyword(s):  
Sperm Cell ◽  
Acrosome Reaction ◽  
Male Gamete ◽  
Inhibitory Effect ◽  
Fertilization In Vitro ◽  
Integrin Receptor ◽  
Male And Female ◽  
Sperm Penetration ◽  
Bovine Spermatozoa

Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm–egg interaction in human. Recently, it has been demonstrated that Fn – when present during bovine IVF – strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (α5β1) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin α5 on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin α5 at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm–oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin α5β1 receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin α5 expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a ‘velcro’ interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm–egg binding.

A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different phosphoglucomutase-specific probes

2000 ◽  
Vol 78 (6) ◽  
pp. 683-690 ◽  
Author(s):  
Elzbieta Wyroba ◽  
Birgit H Satir
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Genomic Dna ◽  
Pcr Amplification ◽  
Molecular Size ◽  
Molecular Probes ◽  
Hybridization Analysis ◽  
Yeast Genome ◽  
Specific Primers ◽  
Genome Database

Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phospho glyco protein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes. These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2. Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3 – I-3) not found in other species. PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe. A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.Key words: parafusin, phosphoglucomutase, yeast, hybridization, PCR.

scholarly journals Identification of a novel lncRNA (G3R1) regulated by GLIS3 in pancreatic β-cells

2020 ◽  
Vol 65 (3) ◽  
pp. 59-67
Author(s):  
David W Scoville ◽  
Artiom Gruzdev ◽  
Anton M Jetten
Keyword(s):  
Pancreatic Islets ◽  
Knockout Mouse ◽  
Cell Function ◽  
Significant Loss ◽  
Messenger Rnas ◽  
Β Cells ◽  
Β Cell ◽  
Pancreatic Β Cells ◽  
Phenotypic Analysis ◽  
Cell Functions

Recent advances in high throughput RNA sequencing have revealed that, in addition to messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs) play an important role in the regulation of many cell functions and of organ development. While a number of lncRNAs have been identified in pancreatic islets, their function remains largely undetermined. Here, we identify a novel long ncRNA regulated by the transcription factor GLIS3, which we refer to as GLIS3 regulated 1 (G3R1). This lncRNA was identified for its significant loss of expression in GLIS3 knockout mouse pancreatic islets. G3R1 appears to be specifically expressed in mouse pancreatic β-cells and in a β-cell line (βTC-6). ChIP-seq analysis indicated that GLIS3 and other islet-enriched transcription factors bind near the G3R1 gene, suggesting they directly regulate G3R1 transcription. Similarly, an apparent human homolog of G3R1 displays a similar expression pattern, with additional expression seen in human brain. In order to determine the function of G3R1 in mouse pancreatic β-cells, we utilized CRISPR to develop a knockout mouse where ~80% of G3R1 sequence is deleted. Phenotypic analysis of these mice did not reveal any impairment in β-cell function or glucose regulation, indicating the complexity underlying the study of lncRNA function.

Non-coding RNA profiling as an indicator of male gamete reproductive potential

2016 ◽  
Vol 106 (3) ◽  
pp. e302
Author(s):  
T. Cozzubbo ◽  
N. Pereira ◽  
S. Cheung ◽  
A.P. Clement ◽  
Z. Rosenwaks ◽  
...  
Keyword(s):  
Reproductive Potential ◽  
Male Gamete ◽  
Rna Profiling ◽  
Non Coding Rna

scholarly journals Predicting Response to Neoadjuvant Therapy in Colorectal Cancer Patients the Role of Messenger-and Micro-RNA Profiling

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1652 ◽  
Author(s):  
Alberto Izzotti ◽  
Chiara Ceccaroli ◽  
Marta Geretto ◽  
Filippo Grillo Ruggieri ◽  
Sara Schenone ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Neoadjuvant Therapy ◽  
Cancer Patients ◽  
Micro Rna ◽  
Molecular Signature ◽  
Messenger Rnas ◽  
Rna Profiling ◽  
Micro Rnas ◽  
Colorectal Cancer Patients ◽  
Study Designs

Colorectal cancer patients’ responses to neoadjuvant therapy undergo broad inter-individual variations. The aim of this systematic review is to identify a molecular signature that is predictive of colon cancer downstaging and/or downgrading after neoadjuvant therapy. Among the hundreds analysed in the available studies, only 19 messenger-RNAs (mRNAs) and six micro-RNAs (miRNAs) were differentially expressed in responders versus non-responders in two or more independent studies. Therefore, a mRNA/miRNA signature can be designed accordingly, with limitations caused by the retrospective nature of these studies, the heterogeneity in study designs and the downgrading/downstaging assessment criteria. This signature can be proposed to tailor neoadjuvant therapy regimens on an individual basis.

Male gamete messenger RNAs inAllomyces

1981 ◽  
Vol 5 (2) ◽  
pp. 173-177
Author(s):  
Arthur Eisenberg ◽  
Joseph P. Mascarenhas
Keyword(s):  
Male Gamete ◽  
Messenger Rnas

scholarly journals PEPPRO: quality control and processing of nascent RNA profiling data

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jason P. Smith ◽  
Arun B. Dutta ◽  
Kizhakke Mattada Sathyan ◽  
Michael J. Guertin ◽  
Nathan C. Sheffield
Keyword(s):  
Fault Tolerant ◽  
Analysis Pipeline ◽  
Web Based ◽  
Rna Integrity ◽  
Rna Profiling ◽  
Project Report ◽  
Library Complexity ◽  
Nascent Rna ◽  
Assess Quality ◽  
Downstream Analysis

AbstractNascent RNA profiling is growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable workflow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniformly processed output files for downstream analysis and assesses adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report. PEPPRO can be run locally or using a cluster, providing a portable first step for genomic nascent RNA analysis.

scholarly journals The Ribosome as a Missing Link in Prebiotic Evolution III: Over-Representation of tRNA- and rRNA-Like Sequences and Plieofunctionality of Ribosome-Related Molecules Argues for the Evolution of Primitive Genomes from Ribosomal RNA Modules

2019 ◽  
Vol 20 (1) ◽  
pp. 140 ◽  
Author(s):  
Robert Root-Bernstein ◽  
Meredith Root-Bernstein
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Relevant Literature ◽  
Messenger Rnas ◽  
Rna Sequences ◽  
Transfer Rnas ◽  
Cell Functions ◽  
Cellular Evolution ◽  
Full Complement ◽  
Reading Frames

We propose that ribosomal RNA (rRNA) formed the basis of the first cellular genomes, and provide evidence from a review of relevant literature and proteonomic tests. We have proposed previously that the ribosome may represent the vestige of the first self-replicating entity in which rRNAs also functioned as genes that were transcribed into functional messenger RNAs (mRNAs) encoding ribosomal proteins. rRNAs also encoded polymerases to replicate itself and a full complement of the transfer RNAs (tRNAs) required to translate its genes. We explore here a further prediction of our “ribosome-first” theory: the ribosomal genome provided the basis for the first cellular genomes. Modern genomes should therefore contain an unexpectedly large percentage of tRNA- and rRNA-like modules derived from both sense and antisense reading frames, and these should encode non-ribosomal proteins, as well as ribosomal ones with key cell functions. Ribosomal proteins should also have been co-opted by cellular evolution to play extra-ribosomal functions. We review existing literature supporting these predictions. We provide additional, new data demonstrating that rRNA-like sequences occur at significantly higher frequencies than predicted on the basis of mRNA duplications or randomized RNA sequences. These data support our “ribosome-first” theory of cellular evolution.

scholarly journals sRNAbench and sRNAtoolbox 2019: intuitive fast small RNA profiling and differential expression

2019 ◽  
Vol 47 (W1) ◽  
pp. W530-W535 ◽  
Author(s):  
Ernesto Aparicio-Puerta ◽  
Ricardo Lebrón ◽  
Antonio Rueda ◽  
Cristina Gómez-Martín ◽  
Stavros Giannoukakos ◽  
...  
Keyword(s):  
Differential Expression ◽  
Rna Sequencing ◽  
Small Rna ◽  
Pcr Amplification ◽  
Small Rna Sequencing ◽  
User Friendliness ◽  
Rna Profiling ◽  
Sequencing Studies ◽  
Microrna Sequencing ◽  
Downstream Analysis

Abstract Since the original publication of sRNAtoolbox in 2015, small RNA research experienced notable advances in different directions. New protocols for small RNA sequencing have become available to address important issues such as adapter ligation bias, PCR amplification artefacts or to include internal controls such as spike-in sequences. New microRNA reference databases were developed with different foci, either prioritizing accuracy (low number of false positives) or completeness (low number of false negatives). Additionally, other small RNA molecules as well as microRNA sequence and length variants (isomiRs) have continued to gain importance. Finally, the number of microRNA sequencing studies deposited in GEO nearly triplicated from 2014 (280) to 2018 (764). These developments imply that fast and easy-to-use tools for expression profiling and subsequent downstream analysis of miRNA-seq data are essential to many researchers. Key features in this sRNAtoolbox release include addition of all major RNA library preparation protocols to sRNAbench and improvements in sRNAde, a tool that summarizes several aspects of small RNA sequencing studies including the detection of consensus differential expression. A special emphasis was put on the user-friendliness of the tools, for instance sRNAbench now supports parallel launching of several jobs to improve reproducibility and user time efficiency.

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