Development of multiplex real-time RT-PCR to determine the expression level of Toll-like receptor genes

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Abstract

Immune response gene expression analysis is an important task in studies of interactions between 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 remain unsolved. In the present work, HPRT1, SDHA, GAPDH and TBP were selected as candidates for reference genes with stable expression and a system for their analysis based on multiplex real-time RT-PCR was developed. The results of calculations using geNorm and BestKeeper algorithms allowed to create a stable index based on two genes — HPRT1 and SDHA. It was used for normalization of expression level of target genes of Toll-like receptors: TLR1, TLR2, TLR4, TLR6 and TLR8. The obtained expression values of Toll-like receptor genes in the sample of conditionally healthy individuals were characterized by high stability and positive mutual correlation (except TLR6), which may indicate common mechanisms of expression regulation and also confirms the possibility of using the developed multiplex system to analyze the expression of immune response genes.

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About the authors

S. A. Salamaikina

Central Research Institute of Epidemiology Federal Service for Surveillance on Consumer Rights Protection and Human Well-Being; Moscow Institute of Physics and Technology, Dolgoprudny

Author for correspondence.
Email: salamaykina@cmd.su
Russian Federation, Moscow; Dolgoprudny, Moscow Region

V. I. Korchagin

Moscow Institute of Physics and Technology

Email: salamaykina@cmd.su
Russian Federation, Dolgoprudny, Moscow Region

K. O. Mironov

Central Research Institute of Epidemiology Federal Service for Surveillance on Consumer Rights Protection and Human Well-Being

Email: salamaykina@cmd.su
Russian Federation, Moscow

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Supplementary files

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2. Fig. 1. Scheme of arrangement of oligonucleotides for detection of target regions and determination of gene expression level. a — Annealing of one of the primers in two exons; b — fragment size exceeds possible processivity of the Taq polymerase used; I — DNA sequence; II — cDNA sequence.

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3. Fig. 2. Schematic diagram of experiments to determine expression levels in two multiplexes.

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4. Fig. 3. Distribution of GM [Ct] values ​​of housekeeping genes.

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5. Fig. 4. Pairwise correlation diagram of GM [Ct] values ​​of housekeeping genes. Statistically insignificant results are marked.

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6. Fig. 5. Distribution of relative expression values ​​(Cq) of TLR genes.

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7. Fig. 6. Pairwise correlation diagram of relative expression values ​​(Cq) of TLR genes.

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