Validation of Reference Gene Stability for Mirna Quantification by Reverse Transcription Quantitative Pcr in the Peripheral Blood of Patients With Covid-19 Critical Illness

Amanda Formosa, Erica Acton, Amy Lee, Paul Turgeon, Shehla Izhar, Pamela Plant, Jim N. Tsoporis, Sabri Soussi, Uriel Trahtemberg, Andrew Baker, Claudia C. dos Santos


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.


The World Health Organization declared the coronavirus disease 2019 (COVID-19) pandemic in March 2020 [1]. As of August 2022, COVID-19 has accounted for more than 577 million confirmed cases and more than six million confirmed deaths [2]. The course of illness of COVID-19 ranges from mild to severe and can be vastly unpredictable [3, 4], however certain groups are particularly vulnerable to severe illness such as the elderly and comorbid patient population [5]. Mortality rates in the literature range from 17–39% among critically ill patients [5]. While the advent of vaccination has had a remarkable effect in preventing serious illness [6, 7], vaccinated individuals can suffer breakthrough infections with high illness severity [4]. Given the variable clinical course of this disease, clinicians face challenges in assessing (i) a patient’s vulnerability and (ii) deciding when and if to initiate appropriate therapeutic interventions.

Materials and method

Approval for the collection of peripheral blood from critically ill patients was obtained from the St. Michael’s Hospital Research Ethics Board (REB number 20–078). Written consent from substitute decision makers was obtained for enrollment in our study as per institution protocol. Blood was collected within 48 hours of ICU admission into PAXgene Blood RNA Tubes (PreAnalytiX, Hombrechtikon, Switzerland, catalog number 762165) as per manufacturer instructions. Briefly, approximately 2.5mL of blood was drawn directly into the PAXgene tube and stored at -80°C until analysis.


NanoString technology was used to profile 798 human miRNAs in the peripheral blood of 24 patients, 12 of which were COVID-19 positive and 12 which were negative.The miRNA expression data was then processed in two distinct manners–Method 1 and Method 2 –so as to obtain an array of candidate reference genes to test in RT-qPCR and major computational programs that analyze reference genes. RT-qPCR/computational analysis was done in a total of 41 patients (of note, this included 16 of the NanoString patients already mentioned). Table 1 lists the top five miRNAs deemed as most stable by each method.


Identifying stable reference genes is of the utmost importance in correctly interpreting gene expression in any experimental design. The choice of a wrong reference gene can greatly affect the target gene results, and the use of multiple reference genes is considered to be important for accurate analysis [33, 50]. The current report sought to identify reliable reference genes for use in RT-qPCR miRNA expression studies in the peripheral blood of critically ill patients with and without COVID-19.


We thank our research coordinators Gyan Sandhu and Dr. Valeria Di Giovanni for their contributions to human sample collection and organizing of our biobank, respectively. We also thank patients that donated samples for this project.

Citation: Formosa A, Acton E, Lee A, Turgeon P, Izhar S, Plant P, et al. (2023) Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness. PLoS ONE 18(8): e0286871.

Editor: Jacopo Sabbatinelli, Universita Politecnica delle Marche, ITALY

Received: February 8, 2023; Accepted: May 25, 2023; Published: August 29, 2023

Copyright: © 2023 Formosa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: A NanoString grant provided this study with reagents for experiments and technical support. The St. Michael’s Hospital Foundation provided financial support to collect patient samples.

Competing interests: The authors have declared that no competing interests exist.

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