Investigating expression of CIB4 gene in different tissues of Kermani Sheep using Real Time qPCR

Kermani sheep is one of the most important Iranian native sheep breeds and is well-adapted to harsh environmental conditions of south-eastern part of country, where dry and hot weather is prevalent and pastures are of low quality and quantity. CIB4 protein is one member of the calcium and integrin- binding proteins family, CIB proteins which are effective in high fertility. This protein has two nucleotides’ sequence including L-CIB4 and S-CIB4. CIB4 biological functions in mammals are not well defined yet, however, a lot attentions has been received to these proteins due to their potential effects in increasing fertility. Considering the importance of this protein in fertility, we examined the expression of this gene in Kermani sheep.
In this study gene expression of CIB4 in brain, heart, lung, kidney, liver, ovary and testis (3 repeats from each tissue) in Kermani sheep (4 males and 2 females) were analyzed. Initially, the various tissues of sheep supplied and in liquid nitrogen, transported to the laboratory and then total RNA from all samples were extracted based on One Step RNA Reagent kit manual and cDNA has been made using cDNA Synthesis Kit of Fermentase Co. After amplifying CIB4 and GAPDH gene, as an internal control, results of Real Time PCR were analyzed and PCR products were separated on 2% agarose gel.
Spectrophotometry results confirmed good quality of extracted RNA and observation of two bands, 28S and 18S on gel electrophoresis of RNA showed that RNA is intact and absence of additional bands represents purity of RNA. Observation of 574 bp fragment for CIB4 gene in testis tissue and absence of this band in other tissues and observation of 150 bp fragments for GAPDH gene in all tissues indicated that this gene is only expressed in testes and in brain, heart, lung, kidney, liver and ovary is not expressed.
According to the results CIB4 gene expressed only in testis tissue of Kermani sheep and in brain, heart, lung, kidney, liver and ovary was not expressed, hence may suggest a role of the gene in ram fertility. Although we indicated that this is a testis-specific gene and express only in the testis, but the mechanisms by what CIB4 expression is regulated need to be elucidated.