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The Scientific Basis of Medicine
Published in John S. Axford, Chris A. O'Callaghan, Medicine for Finals and Beyond, 2023
Chris O'Callaghan, Rachel Allen
Every somatic cell carries an identical set of genes. Housekeeping genes are required for basic cellular functions and are constitutively expressed. Additional subsets of expressed genes determine the phenotype and differentiation state of the cell. Access of transcriptional proteins to the gene promoter will be restricted if it is tightly packed within heterochromatin. CpG islands are stretches of cytosine and guanine found at the 5' end of genes. Methylation of cytosine (C) within CpG islands can prevent a gene from becoming expressed. An extreme form of methylation control is exercised on one of the X chromosome pair in every somatic female cell. This process results in hypermethylation of the inactive chromosome to prevent gene transcription. The field of epigenetics studies how processes such as DNA methylation or the modification of histone proteins in nucleosomes are regulated, how they control gene expression and their influence in health and disease. Epigenetic modification of cell cycle control genes is commonly observed in cancer.
Cancer Informatics
Published in Trevor F. Cox, Medical Statistics for Cancer Studies, 2022
The quantile method attempts to normalise the whole distributions of expression across the arrays. Let there be N expression values on each array. These are ordered for each array. The standard distribution is found by averaging the ordered values across the arrays. So the minimum value of the standard distribution is the mean of the minimum values across the arrays. The second smallest value is the mean of the second smallest values of all the arrays, etc. Then for a particular array, the gene with the minimum expression value has that value replaced by the minimum value of the standard distribution, and so forth. Also house-keeping genes, where the samples should have equal expression of these can be used for normalisation, this could involve non-human genes.
Transcriptionally Regulatory Sequences of Phylogenetic Significance
Published in S. K. Dutta, DNA Systematics, 2019
The genome of each cell in an organism shares the same set of genetic information. However, only “housekeeping” genes are expressed in common. Certain genes are transcribed exclusively in specialized cells and are silent in other tissues. The elucidation of the mechanism that turns on (and off) these tissue-specific genes is a fundamental goal in the molecular study of development and differentiation.
Evaluation of Apoptosis Pathway of Geraniol on Ishikawa Cells
Published in Nutrition and Cancer, 2021
Betül Kuzu, Gökhan Cüce, İlknur Çınar Ayan, Burcu Gültekin, Halime Tuba Canbaz, Hatice Gül Dursun, Zafer Şahin, İlknur Keskin, Sabiha Serpil Kalkan
The RT-qPCR method was used to assess the effects of the IC50 dose of geraniol on the mRNA expression level of the apoptosis-related genes on the Ishikawa cells. The Ishikawa cells were seeded at 5 × 105 cells/well in a 6-well plate and incubated for 24 h. After the incubation time, the cells were treated with the IC50 dose of geraniol for 48 h. Total RNA was isolated from the cells treated with geraniol and untreated cells by a Triazole reagent (VWR, USA) according to the manufacturer’s instructions. After the isolated total RNA samples were treated with the DNase-I enzyme, cDNA synthesis was performed using an iScriptTM cDNA Synthesis Kit (BIO-RAD, USA). Bax, Bcl-2, Caspase-3 (CASP-3), Caspase-8 (CASP-8) and Cytochrome C (CYCS), Fas, TNF-α, TNFR1 and TNFR2 gene expression analyses were performed using the real time-quantitative PCR (RT-qPCR)(Bio-Rad, California, USA) method with respect to the Bright Green (abm, USA) method. The levels of the selected genes’ expressions were normalized to the reference GAPDH housekeeping gene.
A functional genomic approach to identify reference genes for human pancreatic beta cell real-time quantitative RT-PCR analysis
Published in Islets, 2021
Maria Inês Alvelos, Florian Szymczak, Ângela Castela, Sandra Marín-Cañas, Bianca Marmontel de Souza, Ioannis Gkantounas, Maikel Colli, Federica Fantuzzi, Cristina Cosentino, Mariana Igoillo-Esteve, Lorella Marselli, Piero Marchetti, Miriam Cnop, Décio L. Eizirik
Quantitative real-time polymerase chain reaction (qPCR) is a commonly used technique to measure mRNA transcript levels owing to its sensitivity, specificity and fast execution.5 The accurate quantification of the observed changes relies on the effective normalization to one or more reference gene(s), whose expression should not be altered by the experimental condition(s) under evaluation. A unique and universal reference gene has not yet been identified, and therefore gene expression normalization usually depends on genes classified as “housekeeping genes” which, due to their cellular indispensability, are assumed to have stable expression under different experimental conditions. Commonly used reference genes, such as beta actin (ACTB), beta-2-microglobulin (B2M), the 18 S ribosome small subunit (18S rRNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are widely used as normalizers due to their robust expression.6,7 However, their expression varies widely among conditions and cell types,8–11 including pancreatic islets.12,13 An inadequate selection of reference genes – that are up – or down-regulated in parallel with the gene under study – could lead to the misinterpretation of qPCR results and obscure genuine changes.
Orchestrating the expression levels of sperm mRNAs reveals CCDC174 as an important determinant of semen quality and bull fertility
Published in Systems Biology in Reproductive Medicine, 2021
Sellappan Selvaraju, Divakar Swathi, Laxman Ramya, Maharajan Lavanya, Santhanahalli Siddalingappa Archana, Muniandy Sivaram
The present study was conducted to evaluate some of the abundantly expressed spermatozoal mRNAs in predicting bull fertility. Though the fertility rates differed significantly, the sperm functional parameters did not vary between the groups suggesting that the sperm functional attributes are not sufficient to identify the sub-fertile bulls (Selvaraju et al. 2008; Somashekar et al. 2015). This warrants studies at the molecular level to assess the subtle differences that are influencing the sperm function and fertility. The gene expression studies in sperm are invariably associated with many challenges including the selection of housekeeping genes. The choice of housekeeping gene is an important factor for calculating the relative gene expression levels in any cell/tissue. The limited gene expression studies on sperm suggested that the commonly reported housekeeping genes for other cells or tissues were not appropriate for the sperm (Lalancette et al. 2008; Feugang et al. 2010). In some studies, ACTB (Chen et al. 2015), RPLP0 (Arangasamy et al. 2011) and RPL23 (Parthipan et al. 2017) were used as housekeeping genes for expression studies in bull sperm. In the present study, though GAPDH and RPL23 had a similar average standard deviation in ΔCt, considering the compactness of the inter-quartile region and stability values, RPL23 was chosen as a housekeeping gene . This is similar to the findings of the earlier study in neat semen from our laboratory (Parthipan et al. 2017).