Analysis of mRNA transcript levels

The relative mRNA expression levels of genes encoding IgA and IL-6 were quantified in liver samples collected from turkeys 2 days after vaccination against ORT. The protocol of qRT-PCR analysis was described by Ognik et al.²⁴. The liver samples were immediately frozen in liquid nitrogen. Subsequently, RNA was isolated using the GeneMATRIX Universal RNA Purification Kit (Eurx, Gdańsk, Poland), in accordance with the manufacturer’s instructions. The concentration of RNA was measured on a NanoDrop spectrophotometer (Nanodrop, NanoDrop Technologies, Wilmington, DE), and RNA integrity was verified on agarose gel under denaturing conditions. qPCR reactions were performed on a LightCycler 480 II apparatus (Roche Applied Science, CA, USA) using the SG qPCR Master Mix (Eurx, Gdańsk, Poland). Reaction conditions were as follows: (i) initial denaturation (one cycle at 95 °C for 10 min) and (ii) amplification (35 cycles of denaturation at 95 °C for 10 s, primer annealing at 58 °C for 10 s and DNA synthesis at 72 °C for 20 s). Endogenous control genes, β-Actin (ACTB) and Vimentins (VIM), were used to normalize gene expression data. The primers for the target and reference genes used in this study are presented in Table ​Table99.

Genes and primers used in the study.

ACTB: B-actin; VIM: vimentin; IL-6: interleukin 6; IgA: immunoglobulin A.