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Abstract <p>Immune response gene expression analysis is an important task in studies of interactions between a host and an infectious agent. Many approaches to this task have been developed, but despite significant progress, the problem of selecting a single standard for data normalization remains unsolved. In the present work, <i>HPRT1</i>, <i>SDHA</i>, <i>GAPDH,</i> and <i>TBP</i> were selected as candidate reference genes with stable expression, and a system based on multiplex real-time RT–PCR was developed for their analysis. Calculations using the geNorm and BestKeeper algorithms made it possible to create a stable index based on two genes, <i>HPRT1</i> and <i>SDHA.</i> The index was used to normalize the expression levels of the target Toll-like receptor genes (TLRs) <i>TLR1</i>, <i>TLR2</i>, <i>TLR4</i>, <i>TLR6,</i> and <i>TLR8</i>. A high stability and positive mutual correlations were observed for expression values of the TLR genes (except <i>TLR6</i>) in a sample of healthy individuals. The finding suggested common mechanisms of expression regulation and confirmed that the multiplex system is suitable for analyzing expression of immune response genes.</p>

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Development of Multiplex Real-Time RT–PCR to Determine the Expression Levels of Toll-Like Receptor Genes

  • 期刊论文

  • 发表日期：

    2025年2月1日

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  • S. A. Salamaikina,
  • V. I. Korchagin,
  • K. O. Mironov

摘要

Abstract

Immune response gene expression analysis is an important task in studies of interactions between a host and an infectious agent. Many approaches to this task have been developed, but despite significant progress, the problem of selecting a single standard for data normalization remains unsolved. In the present work, HPRT1, SDHA, GAPDH, and TBP were selected as candidate reference genes with stable expression, and a system based on multiplex real-time RT–PCR was developed for their analysis. Calculations using the geNorm and BestKeeper algorithms made it possible to create a stable index based on two genes, HPRT1 and SDHA. The index was used to normalize the expression levels of the target Toll-like receptor genes (TLRs) TLR1, TLR2, TLR4, TLR6, and TLR8. A high stability and positive mutual correlations were observed for expression values of the TLR genes (except TLR6) in a sample of healthy individuals. The finding suggested common mechanisms of expression regulation and confirmed that the multiplex system is suitable for analyzing expression of immune response genes.