Analysis of the stability of the reference genes GAPDH, SDHA and RPL-19 in sheep from a semi-arid region infected by gastrointestinal nematodes

Abstract

Analyzing the stability of reference genes already described as universal is an important methodology to lead gene expression analysis because different studies have shown that the expression of universal reference genes may vary between experimental treatments. In this sense, the glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Succinate dehydrogenase complex subunit A (SDHA) and Ribosomal Protein L-19 (RPL-19) reference genes (already described in other studies with sheep from different regions, breeds and infectious agents or in organisms evolutionarily close to sheep) were investigated in the abomasum, small and large intestines of resistant and susceptible crossbred sheep groups to gastrointestinal nematode infections in the Semi-arid region in Northeast of Brazil. The animals were naturally infected to determine the resistance or susceptibility status by counting eggs per gram (EPG) of feces from the gastrointestinal tract after 33 weeks of observations of infection evolution. Relative gene expression was performed by RT-qPCR methodology using Sybr green and relative gene expression stability was tested by different software programs such as REST, BestKeeper, geNorm and Normfinder. Our results showed the susceptible animals had increase in egg counts per gram of feces than resistant animals (p < 0.001), and both groups showed a mixed infection by nematodes of the genus Haemonchus, Trichostrongylus, Oesophagostomum and Trichuris. Furthermore, we show the importance of analyzing different genes in different software programs and the importance to choose ideal reference genes. In this sense, GAPDH was the most stable gene in the abomasum, whereas SDHA was the most stable in the small and large intestines. In addition, we discuss about variables which can interfere in relative expression such as breed, species, climate and tissue. However, utilizing other reference genes already described in other studies with the same and different variables should be performed.

Keywords: GAPDH; Gastrointestinal tract; Sheep and RT-qPCR.

Fig. 1

Average Egg Per Gram (EPG) of feces of the resistant (R) (n = 6) and susceptible (S) (n = 6) groups obtained from the 33 weeks experimental period. t test with Welch’s correction was used to compare groups. The results are shown as the mean ± SD and were represented by ***p < 0.001

Fig. 2

Distribution of expression levels based on Ct values from each reference gene separated by group (resistant and susceptible) in the three evaluated tissues. No samples indicated a significant difference (p ≤ 0.05) by the t test with Welch’s correction

Fig. 3

Stability test results calculated by three differents software programs (Bestkeeper, geNorm and Normfinder) for the Abomasum (a), Small intestine (b) and Large intestine (c)