Data highlighting housekeeping gene variability during chikungunya virus infection
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 mentions the sequence of primers used in the study. Table 2 demonstrates the efficiency of the primers used, which was determined by CFX Manager Software by plotting the standard curve. The table2 also mentions the Ct values at different dilutions (10-3, 10-4, 10-5) and the slope of standard curve calculated by the software. Table3 contains the Ct values of the uninfected (Control) and infected (Virus) samples and the fold change of different housekeeping genes of three biological replicates. Fig.1 shows the fold change (mentioned in Table 2) in the expression of housekeeping genes during chikungunya virus infection.
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The primer efficiency was calculated by qPCR. The PCR reaction was performed with each primer (Table 1) using 2µl template cDNA. The PCR product was run on agarose gel and the DNA was purified after excising the desired band. The purified DNA was serially diluted and used as template for qPCR using specific primers. The primer efficiency was generated from CFX Manager 3.0 Software. To determine the relative expression of reference genes (GusB, HPRT, Beta Actin, 18S rRNA, GAPDH) during chikungunya virus infection, HEK293 cells with chikungunya virus. The RNA was isolated 48hr post infection which was subjected to cDNA synthesis. The cDNA was used to perform qPCR using specific primers and the Ct values were obtained from CFX Manager Software. The fold change between the uninfected and infected samples were calculated with 2ΔΔCt method. The statistical significance was determined by the p-value.