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

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.