The Scientific Basis of Medicine
John S. Axford, Chris A. O'Callaghan in Medicine for Finals and Beyond, 2023
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.
Basket Trials at the Confirmatory Stage
Zoran Antonijevic, Robert A. Beckman in Platform Trial Designs in Drug Development, 2018
While some companion diagnostics, such as those that look for mutations, may give a binary result, others, such as those that quantify expression of a gene, give continuous results. For those companion diagnostics with a continuous readout, a cutoff must be established between “biomarker positive” and “biomarker negative” patients, another important goal that should be accomplished based on Phase 2 studies before the confirmatory Phase 3 basket trial is initiated (31). Further, gene expression levels are frequently normalized to the expression levels of “housekeeping genes,” i.e., genes whose expression level is believed to be relatively constant. However, these “housekeeping genes” may vary from tissue to tissue, and should be optimized in the laboratory as part of customizing the companion diagnostic assay for each tissue.
Beneficial Lactic Acid Bacteria
K. Balamurugan, U. Prithika in Pocket Guide to Bacterial Infections, 2019
Housekeeping genes encode products essential for cell survival, and therefore, they undergo changes rarely. However, these genes have been reported to evolve much faster than rRNAs; hence, they can be engaged in bacterial identification (Ochman and Wilson 1987). The phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) partial gene sequences can be used as genomic markers alternative to 16S rRNA gene sequences, and they have a higher discriminatory power for LAB. Strains of the same enterococcal species have at least 99% rpoA and 97% pheS gene sequence similarity, whereas different enterococcal species have at maximum 97% rpoA and 86% pheS gene sequence similarity (Naser et al. 2005). The pheS gene sequence analysis provided the highest discrimination for the identification of different species of lactobacilli. pheS provided an interspecies gap, which normally exceeded 10% divergence and an intraspecies variation up to 3%, while rpoA revealed a lower resolution with an interspecies gap normally exceeding 5% and an intraspecies variation up to 2% (Naser et al. 2007). Additionally, the pheS and rpoA genes were successfully used in identification of novel species (Yi et al. 2013; Chang et al. 2015; Kadri et al. 2015).
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).
Potentiating tumor immunity using aptamer-targeted RNAi to render CD8+ T cells resistant to TGFβ inhibition
Published in OncoImmunology, 2018
Yvonne Puplampu-Dove, Tal Gefen, Anugraha Rajagopalan, Darija Muheramagic, Brett Schrand, Eli Gilboa
CD8+ T cells from 8–10 weeks old C57BL/6 spleens, were purified by positive selection using CD8+ microbeads (Miltenyi Biotech). The purified CD8+ T cells were activated at 1million cells/ml in a 96-well plate using 0.5 µg/ml of plate-bound anti-CD3 (145–2C11) and 0.5 µg/ml of soluble anti-CD28 (37.51) antibodies (BD Pharmigen). Cells were treated (in triplicates) with aptamer-siRNA conjugates at 500 nM concentration 3 times, every 6 hours. 24 hours post last treatment, total RNA was isolated using QIAGEN RNeasy mini kit (QIAGEN) and converted to cDNA using a high capacity cDNA reverse transcriptase kit (Applied Biosystems). Pre-designed Taqman probe sets for specific genes of interest as well as internal control genes (β-actin and GAPDH) were used to analyze gene expression by qPCR on a StepOne qPCR device (Applied Biosystems). Gene expression was normalized to control housekeeping genes.
Identification of reliable reference genes for quantitative real-time PCR in ovary and uterus of laying hens under heat stress
Published in Stress, 2019
Hossein Hassanpour, Zahra Aghajani, Shahab Bahadoran, Navid Farhadi, Hasan Nazari, Waranyoo Kaewduangta
Polymerase chain reaction (PCR) techniques, especially real-time quantitative PCR (RT-qPCR), play a principle role in current cellular and molecular biology (Kubista et al., 2006). In RT-qPCR, two points must be considered: (1) the quantification process is composed of standardized procedures, assay quality and calculation methods, and (2) normalization of samples to correct for differences in gene inputs (Dorak, 2007). In this technique, the accurate relative expression of transcripts and their fold changes are estimated by comparison with a reference gene (housekeeping gene) (Wong & Medrano, 2005). In order to function as a reference, the expression of a gene must stay unchanged and constant irrespective of treatment. However, it has been shown that the transcription of these genes is not perfectly unchanged in various types of tissues, cells, phases of disease, and treatment (Gu et al., 2011; Vandesompele, 2002). Hence, determination of a stable housekeeping gene may be important when it is transcribed differentially across the samples (Kozera & Rapacz, 2013; Suzuki, Higgins, & Crawford, 2000).
Related Knowledge Centers
- Cpg Site
- Gene Expression
- Molecular Biology
- Reverse Transcription Polymerase Chain Reaction
- Ribosomal Rna
- Transfer Rna
- Transcription
- Promoter
- Transcription Factor
- Enhancer