scholarly journals 212 GENE EXPRESSION PROFILE OF PROTAMINES AND TRANSITION NUCLEAR PROTEINS IN BOVINE TESTIS

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
M. A. M. M. Ferraz ◽  
R. Simões ◽  
M. P. Milazzotto ◽  
J. A. Visintin ◽  
M. E. O. A. Assumpçäo
Keyword(s):  
Gene Expression ◽  
Rna Extraction ◽  
Great Influence ◽  
Software Tool ◽  
Nuclear Proteins ◽  
Dna Integrity ◽  
Evolutionary Mechanisms ◽  
Relative Expression ◽  
Bovine Sperm ◽  
Commercial Kits

During spermiogenesis, haploid spermatids undergo complex morphological and physiological changes to differentiate into spermatozoa. These processes include chromatin remodelling mediated by the replacement of histones through transition nuclear proteins (Tnp) and protamines (PRM). These proteins have the function to compact and protect the chromatin, exerting great influence on human and mouse fertility. Several studies have demonstrated the positive relationship between unregulated production of protamines and infertility. In humans and mice, PRM1/PRM2 ratio is important to predict fertility. When the 1 : 1 ratio (ideal) is disrupted, in these species, the sperm DNA integrity is altered. Most of the infertility cases caused by protamine deficiency, in humans, are related to PRM2 (Aoki et al. 2005 Hum. Reprod. 20, 1298–1306). Expression levels of PRM2 have been correlated with the DNA damage in mice; however, its role on bull fertility is still unclear. The aim of this study was to determine the relative expression of PRM1, PRM2, PRM3, Tnp1, and Tnp2 in bovine testis. Evaluate the expression of these genes is of utmost importance to understand the role of each protamine during bull spermatogenesis. Testis from post-pubertal bulls (n = 10) were obtained from the slaughterhouse. The RNA extraction and cDNA synthesis were performed using commercial kits. The gene expression (P1, P2, P3, Tnp1, and Tnp2) was determined by real-time RT-PCR using bovine specific primers and β-actin as endogenous controls. A relative expression software tool (Pfaffl et al. 2002 Nucleic Acid Res. 30) was used to compare all samples of each group. The quantification of mRNA relative expression demonstrated a higher expression of PRM1, the relative expression of PRM2 was lower than the relative expression of PRM1 (5.008 ± 1.501 × 23.906 ± 6.174, respectively; P < 0.05). There was no difference between the relative expression of the mRNA for PRM2, Tnp1, and Tnp2 (5.008 ± 1.501, 5.023 ± 1.064, 4.266 ± 1.170, respectively; P > 0.05). The PRM3 mRNA had the lowest relative expression (2.003 ± 0.663). The PRM1/PRM2 ratio found in this study was 4.77 : 1.00. Differently from human and mice, the lower expression of PRM2 mRNA may be an evolutionary adaptation of the bull spermatogenesis, which makes the bovine sperm less susceptible to protamination changes that lead to infertility. More studies are being performed by our research group to evaluate the function of these proteins in bulls. It is fundamental to understand the biology of bovine spermiogenesis, providing knowledge to increase the fertility and be able to elucidate the evolutionary mechanisms that may have caused the possible loss of functionality of PRM2 in bulls. This work was supported financially by FAPESP (09/17035-6 and 07/55294-8).

scholarly journals Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum

2019 ◽  
Vol 43 ◽  
Author(s):  
Rafael Novais de Miranda ◽  
Caroline Marcela da Silva ◽  
Antonio Carlos da Mota Porto ◽  
Welison Andrade Pereira
Keyword(s):  
Gene Expression ◽  
Common Bean ◽  
Sclerotinia Sclerotiorum ◽  
Rna Extraction ◽  
White Mold ◽  
Basic Requirement ◽  
High Quality ◽  
Commercial Kits ◽  
Made In

ABSTRACT The Straw Test is an assay developed to evaluate the resistance of common bean to white mold, in which the plant stems are inoculated and the symptoms of the disease are monitored. It is plausible to admit that investigating gene expression in pathogen-infected tissues may be strategically interesting. However, obtaining a quality RNA is a basic requirement for this purpose. Therefore, the objective of this study was to evaluate adjustments in protocols of commercial kits in the expectation of improving the quality of RNA obtained from bean stems. For this, plants of two lines were inoculated and the stems pathogen-infected were collected 72 hours after. For RNA extraction, two commercial reagents were used following the manufacturer’s recommendations and then following adaptations in these protocols. In particular, the proposed modifications relate to volumes of supernatant recovered in purification steps, additional step of chloroform purification and extended time for nucleic acids precipitation. The obtained RNA was analyzed by spectrophotometer, electrophoresis and bioanalyzer, then converted into cDNA and subsequently submitted to PCR. From the obtained data, it was observed that the adaptations made in the protocols contributed to better results and that, when the indicative values of RNA quality are guaranteed, the subsequent reactions are more pure, precise and representative.

Relative Expression of Mouse Udp-glucuronosyl Transferase 2b1 Gene in the Livers, Kidneys, and Hearts: The Influence of Nonsteroidal Anti-inflammatory Drug Treatment

2019 ◽  
Vol 20 (11) ◽  
pp. 918-923 ◽  
Author(s):  
Yazun Jarrar ◽  
Qais Jarrar ◽  
Mohammad Abu-Shalhoob ◽  
Abdulqader abed ◽  
Esra'a Sha'ban
Keyword(s):  
Gene Expression ◽  
Strongly Correlated ◽  
Chain Reaction ◽  
Inflammatory Drug ◽  
Relative Expression ◽  
Elimination Half ◽  
Endogenous Compounds ◽  
Polymerase Chain ◽  
Anti Inflammatory ◽  
Glucuronosyl Transferase

Background: Mouse Udp-glucuronosyl Transferase (UGT) 2b1 is equivalent to the human UGT2B7 enzyme, which is a phase II drug-metabolising enzyme and plays a major role in the metabolism of xenobiotic and endogenous compounds. This study aimed to find the relative expression of the mouse ugt2b1 gene in the liver, kidney, and heart organs and the influence of Nonsteroidal Anti-inflammatory Drug (NSAID) administration. Methods: Thirty-five Blab/c mice were divided into 5 groups and treated with different commonly-used NSAIDs; diclofenac, ibuprofen, meloxicam, and mefenamic acid for 14 days. The livers, kidneys, and hearts were isolated, while the expression of ugt2b1 gene was analysed with a quantitative real-time polymerase chain reaction technique. Results: It was found that the ugt2b1 gene is highly expressed in the liver, and then in the heart and the kidneys. NSAIDs significantly upregulated (ANOVA, p < 0.05) the expression of ugt2b1 in the heart, while they downregulated its expression (ANOVA, p < 0.05) in the liver and kidneys. The level of NSAIDs’ effect on ugt2b1 gene expression was strongly correlated (Spearman’s Rho correlation, p < 0.05) with NSAID’s lipophilicity in the liver and its elimination half-life in the heart. Conclusion: This study concluded that the mouse ugt2b1 gene was mainly expressed in the liver, as 14-day administration of different NSAIDs caused alterations in the expression of this gene, which may influence the metabolism of xenobiotic and endogenous compounds.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

Oxidants in signal transduction: impact on DNA integrity and gene expression

2005 ◽  
Vol 19 (3) ◽  
pp. 387-394 ◽  
Author(s):  
Kathryn A. Ziel ◽  
Valentina Grishko ◽  
Clayton C. Campbell ◽  
Jeffrey F. Breit ◽  
Glenn L. Wilson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Dna Integrity

scholarly journals Akirins are highly conserved nuclear proteins required for NF-κB-dependent gene expression in drosophila and mice

2007 ◽  
Vol 9 (1) ◽  
pp. 97-104 ◽  
Author(s):  
Akira Goto ◽  
Kazufumi Matsushita ◽  
Viola Gesellchen ◽  
Laure El Chamy ◽  
David Kuttenkeuler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Proteins

scholarly journals NPY and sbGnRH gene expression in juvenile and adult male Brazilian flounder Paralichthys orbignyanus

2011 ◽  
Vol 41 (11) ◽  
pp. 1927-1930 ◽  
Author(s):  
Vinicius Farias Campos ◽  
Tiago Veiras Collares ◽  
Fabiana Kömmling Seixas ◽  
João Carlos Deschamps ◽  
Luis Fernando Fernandes Marins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Extraction ◽  
Sea Bream ◽  
Mrna Levels ◽  
Adult Males ◽  
Adult Fish ◽  
Cdna Synthesis ◽  
Hypothalamic Regulation ◽  
Measure Gene Expression ◽  
Paralichthys Orbignyanus

The objective of this study was to evaluate neuropeptide Y (NPY) and sea bream gonadotropin-release hormone (sbGnRH) gene expression in juvenile and adult males of Brazilian flounder. Hypothalamuses from fish were sampled for total RNA extraction. After cDNA synthesis, real-time PCR was used to measure gene expression. NPY showed approximately 2-fold increases in their mRNA levels while sbGnRH showed 3-fold increases in adult fish. These results suggest that these peptides could be involved on hypothalamic regulation of Brazilian flounder sexual maturation.

scholarly journals TriPOINT: a software tool to prioritize important genes in pathways and their non-coding regulators

2018 ◽  
Vol 35 (15) ◽  
pp. 2686-2689
Author(s):  
Asa Thibodeau ◽  
Dong-Guk Shin
Keyword(s):  
Gene Expression ◽  
Software Tool ◽  
Supplementary Information ◽  
Analysis Tool ◽  
Graph Representations ◽  
Expression Levels ◽  
Conducting Pathway ◽  
Pathway Analysis Tool ◽  
Pathway Analyses ◽  
Gene Expression Levels

Abstract Summary Current approaches for pathway analyses focus on representing gene expression levels on graph representations of pathways and conducting pathway enrichment among differentially expressed genes. However, gene expression levels by themselves do not reflect the overall picture as non-coding factors play an important role to regulate gene expression. To incorporate these non-coding factors into pathway analyses and to systematically prioritize genes in a pathway we introduce a new software: Triangulation of Perturbation Origins and Identification of Non-Coding Targets. Triangulation of Perturbation Origins and Identification of Non-Coding Targets is a pathway analysis tool, implemented in Java that identifies the significance of a gene under a condition (e.g. a disease phenotype) by studying graph representations of pathways, analyzing upstream and downstream gene interactions and integrating non-coding regions that may be regulating gene expression levels. Availability and implementation The TriPOINT open source software is freely available at https://github.uconn.edu/ajt06004/TriPOINT under the GPL v3.0 license. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Relative expression of mRNAs related to cavitation process in bovine embryos produced in vivo and in vitro

2011 ◽  
Vol 40 (1) ◽  
pp. 124-128
Author(s):  
Sabine Wohlres-Viana ◽  
Mariana Cortes Boite ◽  
João Henrique Moreira Viana ◽  
Marco Antonio Machado ◽  
Luiz Sérgio de Almeida Camargo
Keyword(s):  
Culture System ◽  
Rna Extraction ◽  
Endogenous Control ◽  
Bovine Embryos ◽  
Relative Expression ◽  
Gapdh Gene ◽  
Expression Of Genes ◽  
In Vitro Culture System

The objectives of this work were to identify and to evaluate possible differences on gene expression of aquaporins and Na/K-ATPases transcripts between embryos in vivo and in vitro produced. For each group, 15 blastocysts distributed in three pools were used for RNA extraction followed by amplification and reverse transcription. The resulting cDNAs were submitted to Real-Time PCR, using the GAPDH gene as endogenous control. It was not possible to identify AQP1 transcripts. Relative expression of AQP3 (1.33 ± 0.78) and AQP11 (2.00 ± 1.42) were not different in blastocysts in vitro and in vivo produced. Na/K-ATPase α1 gene (2.25 ± 1.07) was overregulated whereas Na/K-ATPase β2 transcripts 0.40 ± 0.30) did not differ among blastocysts produced in vitro from those produced in vivo. Transcripts for gene AQP1 are not present in bovine blastocysts. In vitro culture system does not alter expression of genes AQP3, AQP11 and Na/K-ATPase β2 genes, however, it affects expression of Na/K-ATPase α1.

scholarly journals A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas)

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3840 ◽  
Author(s):  
Ming-An Tsai ◽  
I-Hua Chen ◽  
Jiann-Hsiung Wang ◽  
Shih-Jen Chou ◽  
Tsung-Hsien Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
Reference Genes ◽  
Animal Health ◽  
Rna Extraction ◽  
Disease Onset ◽  
Delphinapterus Leucas ◽  
Amplification Efficiency ◽  
Beluga Whales ◽  
Qrt Pcr

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

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