The validation of housekeeping genes as a reference in quantitative Real Time PCR analysis: application in the milk somatic cells and frozen whole blood of goats infected with caprine arthritis encephalitis virus

Abstract

The validation of housekeeping genes (HKGs) for normalization of RNA expression in Real-Time PCR is crucial to obtain the most reliable results. There is limited information on reference genes used in the study of gene expression in milk somatic cells and the frozen whole blood of goats. Thus, the aim of this study was to propose the most stable housekeeping genes that can be used as a reference in Real-Time PCR analysis of milk somatic cells and whole blood of goats infected with caprine arthritis encephalitis virus (CAEV). Animals were divided into two groups: non-infected (N=13) and infected with CAEV (N=13). Biological material (milk somatic cells and whole blood) was collected 4 times during the lactation period (7, 30, 100 and 240days post-partum). The expression levels of candidate reference genes were analyzed using geNorm and NormFinder software. The stability of candidates for reference gene expression was analyzed for CAEV-free (control) and CAEV-infected groups, and also for both groups together (combined group). The stability of expression of β-actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), cyclophilin A (PPIA), RNA18S1, ubiquilin (UBQLN1) and ribosomal protein large subunit P0 (RPLP0) was determined in milk somatic cells, while ACTB, PPIA, RPLP0, succinate dehydrogenase complex subunit A (SDHA), zeta polypeptide (YWHAZ), battenin (CLN3), eukaryotic translation initiation factor 3K (EIF3K) and TATA box-binding protein (TBP) were measured in frozen whole blood of goats. PPIA and RPLP0 were considered as the most suitable internal controls as they were stably expressed in milk somatic cells regardless of disease status, according to NormFinder software. Furthermore, geNorm results indicated the expression of PPIA/RPLP0 genes as the best combination under these experimental conditions. The results of frozen whole blood analysis using NormFinder software revealed that the most stable reference gene in control, CAEV-infected and combined groups is YWHAZ, and - according to the geNorm results - the combined expression of PPM/YWHAZ genes is the best reference in the presented experiment. The usefulness in gene expression analysis of whole blood samples frozen immediately in liquid nitrogen and stored at -80°C was also proved.

Keywords: Blood; CAEV; Goats; Housekeeping genes; Milk somatic cells; Normalization; Real-Time PCR.