Selection of Suitable Internal Control Gene for Analyzing Innate and Humoral Immune Responses in COVID-19 Gene Expression Studies

Background and Objectives: Gene expression analysis is crucial in order to develop new diagnostic and prognostic markers. Selection of internal control gene is critical step for accurate assessment of quantitative real time PCR (qRT-PCR) results. The accuracy of gene analysis depends on accurate normalization of mRNA fractions using well-expressed genes. This method compensates for errors caused during experimental design. These genes are called internal control or housekeeping genes. Depending upon their constitutive expression at relatively high levels housekeeping genes have traditionally been used for normalization, although often without any proper validation. Fewer studies have been conducted to estimate the suitable housekeeping genes for the expression studies of novel corona virus. A case control study was conducted to identify suitable internal control gene for covid-19 gene expression analysis and to use selected internal control gene to better understand the cross talk between cytokines mediated immune response in host infected by novel corona virus. Materials and Methods: Expression of internal control genes GAPDH and SDHA using qRT-PCR was analyzed for 100 healthy controls and 136 COVID-19 confirmed cases. Statistical algorithms such as Bestkeeper, GeNorm, Delta CT and comprehensive gene stability approaches were used for the selection of suitable internal control gene against 12 genes of innate and immune host response (interferon $\alpha$ & $\gamma$, Interleukin-1 $\alpha, 1-\beta, 4,6,7,10,11,13,15$, and 27) in SARS CoV-19 infection. Results: GAPDH was found to be significantly more consistent in expression as compared to SDHA both in case group and control group $(p < 0.001)$. Statistical approaches have shown stable expression of GAPDH as well with M value 1.1. Conclusions: Our data revealed a notable difference in the expression levels between internal controls (GAPDH & SDHA) in both COVID-19 patients and healthy individuals. This underlines the necessity for thorough validation of internal controls prior to their utilization in quantitative assays.

I. Introduction

Real-time quantitative reverse transcription PCR (RT-qPCR) is one of the most sensitive, specific and high throughput technique to evaluate the transcript levels of a gene, however accuracy of this technique depends upon various factors such as RNA integrity, initial sample quantity and efficiency of reverse transcription. An ideal reference gene is employed to normalize the effect of such factors. Careful selection of reference gene is essential for proper normalization of RT-PCR results [1]. It is recommended to validate internal control genes to determine that the expression remains unaffected by the given experimental treatment [2]. Selection of internal control gene is a critical step for accurate assessment of quantitative real time PCR (qRT-PCR) results. Housekeeping genes or internal controls are considered as a constitutive expressed genes or promotor elements in each cell, however the use of these housekeeping genes in molecular biology experiment relies on the assumption that the level of expression remain same throughout the experiment from cell to cell, sample to sample and treatment to treatment. Though all the experiments need to be validated for its accuracy yet the initial step of normalizing the data is the pivotal stage in validation of an experiment specially if the internal control is the sole arbitrator of the assay.