Data highlighting housekeeping gene variability during chikungunya virus infection

Published: 23 June 2022| Version 1 | DOI: 10.17632/znwx5skkhd.1
Contributors:
Nishtha Agrawal,
Madhu Khanna,
Gagan Dhawan

Description

The selection of a suitable reference gene for the normalization of quantitative PCR data is the most crucial and challenging aspect of gene expression analysis. The data set represents the effect of chikungunya virus infection on the expression of five classical internal reference genes GusB, Beta-actin, HPRT, 18S rRNA and GAPDH. These genes are a part of crucial cellular processes. Usually, these genes demonstrate minimum variation in their expression unless some external stress is applied to the cell. Due to this characteristic, they are commonly used as reference genes for normalizing qPCR data. Hence, the objective of the study is to identify the suitable reference gene for gene expression studies during chikungunya virus infection. Table1 demonstrates the qPCR efficiency of the primers used, which was determined by plotting the standard curve. The efficiency of all the primers was observed to be >90%. Table2 contains the Ct values of the uninfected (Control) and infected (Virus) samples of different housekeeping genes, the average of the triplicate of three biological replicates. Table3 mentions the sequence of primers used in the study. Fig.1 shows the modulation in the expression of housekeeping genes during chikungunya virus infection; GAPDH was found to be most consistent among the different genes with no significant change at the transcriptional level. The results demonstrate a significant variation in the expression of four genes, GusB, Beta-actin, HPRT, and 18SrRNA, in response to chikungunya virus infection. The variation in these genes, which otherwise shows slight variation, suggests their involvement in viral pathogenesis. This could be because the variation in their expression may affect their underlying mechanism as they are a part of critical cellular processes. Another essential piece of information generated from the data highlights the stable expression of GAPDH; hence this could be the most suitable reference gene for the normalization of qPCR data during chikungunya virus infection. Since it is strongly recommended to validate the stable housekeeping gene before data normalization, the present data may serve as a reference for the researcher working on expression studies related to chikungunya virus infection. To the best of our knowledge, this is the first study validating the stable reference gene for the normalization of qPCR data during chikungunya virus infection.

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HEK293 cells with chikungunya virus (MOI I) and reported the relative gene expression profile of five commonly used housekeeping genes: Glucuronidase Beta (GusB), Beta-actin, Hypoxanthine phosphoribosyltransferase (HPRT), 18S ribosomal RNA (18S rRNA) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The candidate cells were infected with the virus, followed by RNA isolation at 48hr post-infection. The isolated RNA was subjected to cDNA synthesis, followed by quantitative PCR analysis of the gene expression. The data was acquired by Real-time PCR machine (CFX96 from Bio-Rad) and the data was analysed by Bio-Rad CFX manager software.

Institutions

Vallabhbhai Patel Chest Institute, University of Delhi

Categories

Virology

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