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. 2025 Jul 22;15(1):26636.
doi: 10.1038/s41598-025-04062-9.

Identification of SnRNA U6 as an endogenous reference gene for normalization of MiRNA expression data in COVID-19 patients

Íris Terezinha Santos de Santana Silva  1   2 , Hllytchaikra Ferraz Fehlberg  2   3 , Fabrício Barbosa Ferreira  1   2 , Murillo Ferreira da Silva  1   2 , Pérola Rodrigues Dos Santos  2   4 , Vinicius Moreira Porto  2 , João Carlos Teixeira Dias  1 , George Rego Albuquerque  2 , Ana Paula Melo Mariano  2 , Sandra Rocha Gadelha  2   4 , Carla Martins Kaneto  5
Affiliations

Identification of SnRNA U6 as an endogenous reference gene for normalization of MiRNA expression data in COVID-19 patients

Íris Terezinha Santos de Santana Silva et al. Sci Rep. .

Abstract

SARS-CoV-2, the virus responsible for COVID-19, exhibits structural differences compared to other coronaviruses that impact transmission dynamics and host response. Although vaccines have reduced severe hospitalizations and deaths, understanding the infection remains challenging, and finding viable biomarkers for disease severity would be beneficial. In this context, microRNAs (miRNAs) are a set of small non-coding RNAs that have emerged as potential biomarkers for numerous diseases. However, assessing miRNA expression requires the normalization of RT-qPCR data using appropriate endogenous reference genes. The selection of these reference genes remains a major challenge, directly impacting the accuracy of expression analyses. Although several small RNAs, including snRNAs, have been evaluated, there is still no consensus regarding the optimal reference genes for the normalization of plasma miRNA expression data in COVID-19 infection. This study evaluated five candidate genes-including RNU6B, snRNA U6 (small nuclear RNAs) and three miRNAs (miR-320a, miR-342-3p, and miR-328) as potential endogenous normalizers for analyzing miRNA expression in the plasma of COVID-19 patients. The snRNA U6 showed greater stability using a combination of statistical algorithms (NormFinder, RefFinder, BestKeeper, and GeNorm), indicating that this snRNA can be used as a robust internal reference for analysis in the plasma of COVID-19 patients.

Keywords: Covid-19; Normalization; RT-qPCR; SARS-CoV-2; miRNA; snRNAU6.

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Conflict of interest statement

Declarations. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of endogenous normalizers stability using NormFinder algorithm.
Fig. 2
Fig. 2
Assessment of endogenous reference gene stability using the different algorithms: BestKeeper, ΔCt, GeNorm and NormFinder.
Fig. 3
Fig. 3
Comparative expression levels of normalization candidates in case individuals with SARS-CoV-2 RNA detectable by RT-qPCR and respiratory symptoms and control (individuals with SARS-CoV-2 RNA undetectable by RT-qPCR groups.
Fig. 4
Fig. 4
Map of Brazil and the state of Bahia, highlighting the area (cities in the southern micro-region) attended by LAFEM/UESC as campaign laboratory to SARS-CoV-2 detection.
Fig. 5
Fig. 5
Flow diagram of the development research processes.
Fig. 6
Fig. 6
Procedures and main steps of miRNA expression analyses by RT-qPCR.
See this image and copyright information in PMC

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