Tropical Medicine & Surgery is an open access publication that features scientific works of considerable significance and impact in all aspects of Tropical Medicine and Surgery: Emerging Infectious Diseases, Neglected Tropical Diseases, Non communicable Diseases, Tropical Biomedicine, Tropical Diseases, Tropical Fish Diseases, Tropical Fish Medicine, Tropical Health Nutrition, Tropical Medicine, Tropical Medicine and Health and Tropical Medicine and Hygiene. The scope of the journal is not just restricted to Tropical Medicine; works related to parasitology, veterinary medicine, Epidemiology which is closely associated with Tropical Medicine Research are equally welcome. It is a boon for researchers and students who wish to be kept abreast of the latest advancements in the field of medicine, it would help them build on the current know-how.

For real-time quantitative PCR (qRT-PCR) to be precise and reliable, reference genes must be carefully chosen. However, no work has looked at possible reference gene expression stability to assess whether or not they are appropriate for use as internal controls in SARS-CoV-2 infection or COVID-19-associated mucormycosis (CAM). We examined the expression stability of the nine most widely used housekeeping genes, including TATA-box binding protein (TBP), cyclophilin (CypA), -2-microglobulin (B2M), 18S rRNA (18S), peroxisome proliferator-activated receptor gamma (PPARG) coactivator 1 alpha (PGC-1), glucuronidase beta (GUSB), hypoxanthine phosphoribosyltrans

We chose the best reference gene using statistical methods (NormFinder, BestKeeper, GeNorm, and RefFinder), and we found that clinical severity has a significant impact on the expression stability of

IMPORTANCE:

To provide new diagnostic, therapeutic, and prognostic methods, gene expression investigations are essential. However, data normalisation with a reference gene, whose expression does not change throughout various illness stages, is necessary for reliable expression determination.

Results can be unreliable if a reference gene is wrongly identified. Unfortunately, not a single study has examined the expression stability of housekeeping genes across the disease spectrum to establish their eligibility as internal controls, despite the impact of COVID-19 on the entire world and the urgent unmet need for better treatments. According to our research, CypA and TBP are the two reference genes for COVID-19 and CAM that are most appropriate. Furthermore, GAPDH, the reference gene that was most frequently employed in COVID-19 investigations, proved to be the least effective. This research closes a significant gap in the field and should make it easier to determine how genes are expressed accurately, which will spur the creation of new molecular diagnostics and medicines for better patient care.

To develop diagnostic, therapeutic, and predictive techniques against infectious pathogens, an understanding of changes in gene expression is essential. Real-time quantitative PCR (qRT-PCR), which has a high sensitivity, specificity, and wide quantification range, is frequently chosen in this situation to monitor gene expression. An endogenous control or reference gene from the same sample is necessary for qRT-PCR, nevertheless, in order to accurately quantify gene expression.

In addition to having a sufficient and constant expression, a good reference gene should also have the least amount of expression variability possible between samples and during experiments. However, the reference genes may frequently exhibit substantial expression variability,impairing results and potentially yielding false positives. Therefore, it is crucial to validate reference genes in each new experimental system.

Due to their steady and constitutive expression, housekeeping genes frequently qualify as reference genes in qRT-PCR experiments. The most often used reference genes or internal controls for qRT-PCR analysis are housekeeping genes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine guanine phosphoribosyl transferase 1 (HPRT-1), cyclophilin A (CypA), ribosomal protein L13A (RPL13A), and -ACTIN. Popular internal controls for viral infections, such as COVID-19, include -ACTIN, GAPDH, and 18S rRNA (18S).