Selection of Ideal Reference Genes for Gene Expression Analysis in COVID-19 and Mucormycosis

Selection of reference genes during real-time quantitative PCR (qRT-PCR) is critical to determine accurate and reliable mRNA expression. Nonetheless, not a single study has investigated the expression stability of candidate reference genes to determine their suitability as internal controls in SARS-CoV-2 infection or COVID-19-associated mucormycosis (CAM). Using qRT-PCR, we determined expression stability of the nine most commonly used housekeeping genes, namely, TATA-box binding protein (TBP), cyclophilin (CypA), β-2-microglobulin (B2M), 18S rRNA (18S), peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PGC-1α), glucuronidase beta (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), β-ACTIN, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in patients with COVID-19 of various severities (asymptomatic, mild, moderate, and severe) and those with CAM. We used statistical algorithms (delta-C [threshold cycle], NormFinder, BestKeeper, GeNorm, and RefFinder) to select the most appropriate reference gene and observed that clinical severity profoundly influences expression stability of reference genes. CypA demonstrated the most consistent expression irrespective of disease severity and emerged as the most suitable reference gene in COVID-19 and CAM. Incidentally, GAPDH, the most commonly used reference gene, showed the maximum variations in expression and emerged as the least suitable. Next, we determined expression of nuclear factor erythroid 2-related factor 2 (NRF2), interleukin-6 (IL-6), and IL-15 using CypA and GAPDH as internal controls and show that CypA-normalized expression matches well with the RNA sequencing-based expression of these genes. Further, IL-6 expression correlated well with the plasma levels of IL-6 and C-reactive protein, a marker of inflammation. In conclusion, GAPDH emerged as the least suitable and CypA as the most suitable reference gene in COVID-19 and CAM. The results highlight the expression variability of housekeeping genes due to disease severity and provide a strong rationale for identification of appropriate reference genes in other chronic conditions as well. IMPORTANCE Gene expression studies are critical to develop new diagnostics, therapeutics, and prognostic modalities. However, accurate determination of expression requires data normalization with a reference gene, whose expression does not vary across different disease stages. Misidentification of a reference gene can produce inaccurate results. Unfortunately, despite the global impact of COVID-19 and an urgent unmet need for better treatment, not a single study has investigated the expression stability of housekeeping genes across the disease spectrum to determine their suitability as internal controls. Our study identifies CypA and then TBP as the two most suitable reference genes for COVID-19 and CAM. Further, GAPDH, the most commonly used reference gene in COVID-19 studies, turned out to be the least suitable. This work fills an important gap in the field and promises to facilitate determination of an accurate expression of genes to catalyze development of novel molecular diagnostics and therapeutics for improved patient care.