qRT-PCR data normalization by the identification of expression analysis of the most stable and the least stable housekeeping genes (HKGs) in Covid-19
Keywords:
Housekeeping genes (HKGs), Covid-19, gene expression, expression levels, vaccinesAbstract
Housekeeping genes (HKGs) are known as constitutive and metabolic genes which remain functionally active throughout the life span of an organism. The selection of appropriate housekeeping genes (HKGs) is the prime step for qRT-PCR data normalization. In present study, it was focused to compare the stability of ten HKGs systematically to identify the most stable endogenous internal control for our study groups i.e. control Covid-19 groups (healthy candidates), and Covid-19 effected group (mild, moderate severe). Expression levels of GAPDH, β -actin, 18S rRNA, TBP, RPL 13a, EEF1G, UBE2D2, HPRT1 have determined by using three different biostatical based applets, geNorm, NormFinder, and BestKeeper. For comparative analysis, IL-10 gene was selected as target gene. The results suggested that B2M showed significant expression variation and graded as the least stable housekeeping genes (HKGs). However, beta-Actin, and TBP were identified as the most stable housekeeping genes (HKGs) to carry out genetic based expression analysis of all samples associated with Covid-19. This study has applied the knowledge of molecular biology, immunological, pathological, and bioinformatics in an integrated manner to pave the ways for SARS-CoV-2 transcriptome analysis for the development of effective vaccines against Covid-19 in the future.
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