Real time-qPCR is the most reliable method for evaluation of mRNA expression levels. However, to obtain accurate results, selection of suitable reference genes is necessary for normalizing the real-time qPCR data. The aim of this research was to validate the expression stability of three potential reference genes (ACTB, GAPDH and UXT) in milk somatic cells of Holstein dairy cattle under different lactation stages. For this purpose two types of milk samples from eighteen healthy cows at three lactation stages (early, middle and late of lactation cycle) and four mastitic cows were included in this experiment. Total RNA was extracted from the milk somatic cells and then cDNA was synthesized. Real-time polymerase chain reaction (PCR) performed for actb, gapdh and UXT genes as candidate reference genes. Then, the real-time PCR results were analyzed with BestKeeper program. The evaluation of selected genes by real-time PCR revealed that all genes were expressed in the healthy and mastitic dairy cows. In addition, the UXT and GADPH genes displayed the lowest and highest values of expression level, respectively. The ACTB gene was considered as the most suitable internal controls as it was stably expressed in milk somatic cells regardless of dairy cows conditions. Taken together, our results could help to select suitable reference gene for the normalization of expression levels in milk somatic cells of dairy cattle. تفاصيل المقالة

Growth differentiation factor 9 (GDF9) belong to the superfamily of transforming growth factor β that is highly expressed in growing ovarian follicles of oocyte, and it has been strongly related to fecundity traits in sheep. Therefore, the GDF9 gene could serve as a genetic marker for improvement of reproductive performance in sheep. Therefore, the aim of this study was to investigate the fecundity gene of GDF9 in two local sheep breeds, Bahmaei and Lak Ghashghaei, reared at Kuhgiloyeh and Boyer-Ahmad province of Iran. For this purpose, genomic DNA was extracted from 20 Bahmaei and 20 Lak Ghashghaei (10 single birth, 10 twin births in each breed) multiparous ewes. The entire length of exon 1 and 2 in ovine GDF9 gene was amplified using three designed primer pairs, and detection of single nucleotide polymorphisms (SNPs) in the amplified fragments was determined by direct DNA sequencing method. The first and third amplified fragments of ovine GDF9 showed single genotype in two breeds. Sequencing results of the second fragment (P2) discovered 4 SNPs, including 3 SNPs in coding sequence of exon 2 and one novel in intron of GDF9 gene. No significant association was obtained between combined genotypes of identified SNPs with litter size in both sheep breeds. In general, the two sheep breeds were different in their genetic structure with regard to the GDF9 gene. Frequency distributions of identified SNPs in exon 2 GDF9 gene were different in two sheep breeds. تفاصيل المقالة