Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness

The COVID-19 pandemic has created an urgency to study the host gene response that leads to variable clinical presentations of the disease, particularly the critical illness response. miRNAs have been implicated in the mechanism of host immune dysregulation and thus hold potential as biomarkers and/or therapeutic agents with clinical application. Hence, further analyses of their altered expression in COVID-19 is warranted. An important basis for this is identifying appropriate reference genes for high quality expression analysis studies. In the current report, NanoString technology was used to study the expression of 798 miRNAs in the peripheral blood of 24 critically ill patients, 12 had COVID-19 and 12 were COVID-19 negative. A list of potentially stable candidate reference genes was generated that included ten miRNAs. The top six were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a total of 41 patients so as to apply standard computational algorithms for validating reference genes, namely geNorm, Norm- Finder, BestKeeper and RefFinder. There was general agreement among all four algorithms in the ranking of four stable miRNAs: miR-186-5p, miR-148b-3p, miR-194-5p and miR-448. A detailed analysis of their output rankings led to the conclusion that miR-186-5p and miR- 148b-3p are appropriate reference genes for miRNA expression studies using PaxGene tubes in the peripheral blood of patients critically ill with COVID-19 disease.