Identification of appropriate reference genes for local immune-related studies in Morada Nova sheep infected with Haemonchus contortus

Abstract

Due to the great economic impact of Haemonchus contortus on sheep farming, there is an increasing number of studies addressing host resistance against this nematode, including identification of directly related immune mechanisms. In this context, relative gene expression by RT-qPCR have been largely used, due to its rapidity, high sensitivity, specificity, and reproducibility. Although, appropriate reference gene selection is crucial for accurate interpretation of results. In this study, five reference genes (GAPDH, G6PDH, YWHAZ, ACTB, and B2M) were tested for expression stability in abomasum (fundic and pyloric regions) and abomasal lymph nodes of Morada Nova sheep classified as resistant (n = 5) or susceptible (n = 5) to H. contortus infection in a flock of 151 animals. GAPDH combined with YWHAZ were selected as reference genes for abomasal fundic region and abomasal lymph nodes, whereas YWHAZ was the most stable gene for abomasal pyloric region. These genes presented the lowest intra- and inter-group variations and, consequently, highest stability. In contrast, expression of G6PDH was the least stable in all tissues. The impact of reference gene selection was demonstrated by relative quantification of a pro-inflammatory cytokine (TNFα) in abomasal fundic region. Significant differences in TNFα expression levels between resistant and susceptible groups were only observed when the most stable genes (GAPDH combined with YWHAZ) or GAPDH were used as reference genes, whereas no significant differences were observed when other tested reference genes were used. It was demonstrated that normalization of expression data using inappropriate reference genes may significantly influence interpretation results.

Local and systemic immune mediators of Morada Nova lambs with divergent Haemonchus contortus resistance phenotypes

“…Gene expression results for each target gene were normalized by the reference gene YWHAZ (abomasal pyloric mucosa) or by the geometric mean of GAPDH and YWHAZ (abomasal fundic mucosa and abomasal lymph nodes), as described in a previous study 34 . For blood samples, the gene expression results were normalized by YWHAZ .…”

Innate Immune Responses Associated with Resistance againstHaemonchus contortusin Morada Nova Sheep

“…The relative gene expression was normalized by the reference gene YWHAZ (abomasal pyloric mucosa) and by the geometric mean of the Cq values of GAPDH and YWHAZ (abomasal fundic mucosa and abomasal lymph nodes), according to the stability test previously performed [38]. For the blood samples, five different genes ( GAPDH , G6PDH , YWHAZ , ACTB , and B2M ) were tested with the programs BestKeeper, NormFinder, and RefFinder, which identified YWHAZ as the most stable reference gene.…”

“…Previously reported reference gene stability test data were used to support this study and are available at 10.1007/s11033-018-4281-x. These prior study is cited at relevant places within the text as reference [38]. All remaining data are included within the article.…”

Resistance to Haemonchus contortus in Corriedale sheep is associated to high parasite-specific IgA titer and a systemic Th2 immune response

“…Gastrointestinal nematode infections are one of the main causes of economic losses to ovine farmers worldwide. Among nematodes, Haemonchus contortus is considered one of the most pathogenic and most economically important infectious agents 1 . The parasite, a hematophagous trichostrongyle nematode, causes severe anaemia, hypoproteinemia and submandibular edema in infected sheep that can lead to death depending on the parasite load 2 .…”