Key result
Multiplex PCR system detects mRNA changes in mouse lung immune sensors 24 hours post-influenza infection.
Why the study?
Intracellular pattern recognition receptors are critical for innate antiviral protection, requiring a quantitative PCR system to assess TLR3, TLR7, TLR8, TLR9, MDA5, and RIGI gene expression in mouse tissues and organs.
A newly developed multiplex PCR system successfully quantified significant changes in endosomal and cytosolic receptor mRNA levels in the lungs of mice 24 hours after influenza A infection.
Answers are grounded in this paper and cite their sources.
Extends validated tools for profiling innate receptor expression in murine influenza models; leaves open human translation and clinical utility.
BACKGROUND: The innate immune response plays a crucial role in protecting the organism against viral pathogens, important part of which are pattern recognition receptors, such as Toll-like and RIG-I-like receptors. It is known that viral invasion, including influenza virus infection, leads to the activation of intracellular pattern recognition receptors such as TLR3, TLR7, TLR8, and TLR9, which are localized in the endoplasmic reticulum, endosomes, and lysosomes, as well as MDA5 and RIG-I, which are cytosolic sensors of viral RNA not associated with cell membranes. The expression of these genes, their proper functioning, and regulation are of critical importance for ensuring an adequate immune response and the establishment of antiviral protection. AIM: The aim of this study is to develop and validate a quantitative PCR system for assessing the expression of TLR3, TLR7, TLR8, TLR9, MDA5, and RIGI genes in mouse tissues and organs. METHODS: Gene expression levels were analyzed using reverse transcription polymerase chain reaction with specially developed panels of primers and fluorescent probes. For approbation were selected female inbred BALB/c albino mice aged 8–10 weeks, infected with influenza A/PR8/34 (H1N1) virus. RESULTS: In this study, a test system based on multiplex polymerase chain reaction was developed for assessing the expression of endosomal receptor genes TLR3, TLR7, TLR8, and TLR9, as well as cytosolic sensors MDA5 and RIG-I. The amplification efficiency was 99 for TLR3, 106 for TLR7, and 107% for the remaining genes. This test system was used to study the expression levels of TLRs and RLRs in the lung and spleen tissues of BALB/c mice infected with influenza A/PR8/34 (H1N1) virus. According to the obtained results, 24 hours post-infection, a significant change in mRNA levels of TLR3, TLR7, TLR8, TLR9, and MDA5 was observed in the lungs but not in the spleens of infected animals. CONCLUSION: The developed test system can be used for analyzing the expression of certain intracellular PRRs, providing opportunities for a deeper investigation of the pathophysiological mechanisms underlying the immune response.
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Oleynik et al. (2025) studied Influenza A/PR8/34 (H1N1) virus infection. Multiplex quantitative PCR system was evaluated on Expression levels of TLR3, TLR7, TLR8, TLR9, MDA5, and RIG-I genes. A multiplex PCR test system was developed with 99-107% amplification efficiency, detecting significant changes in mRNA levels of endosomal receptors and cytosolic sensors in mouse lungs 24 hours post-influenza infection.
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