Characterization of COX-2 gene using RNA based technique in endometrial epithelial cells of buffalo (Bubalus bubalis)

The present study has been reported towards molecular characterization of COX-2 gene (PTGS2 gene) in buffalo endometrium. Non-pregnant buffalo uteri were collected from the local abattoir immediately after slaughter and total RNA was extracted. First strand cDNA was synthesized from approximately 2 μg total RNA using iScript cDNA synthesis kit. Following cDNA synthesis, PCR amplification of cDNA was carried out using COX-2 gene specific primers. To determine the optimum conditions, different concentrations of MgCl2, template DNA, Taq DNA polymerase, primers, dNTPs as well as different cycling programmes were analysed. Reproducible amplification pattern was obtained using 2 μl cDNA template (50 ng/μl), 2.5 μl 10X Taq Buffer, 0.5 μl dNTP (50 mM), 2.5 μl MgCl2 (25 mM), 0.5 μl each primer (10 pM/μl), 0.1 μl Phusion DNA polymerase (5 U/μl) and nuclease-free water. The purified PCR amplified product (449 bp) was sequenced using COX-2 gene specific primers. Nucleotide sequence analysis of PCR amplified product exhibited 100 percent identity with the reference sequence of Bubalus bubalis COX-2 gene using Clustal Omega Multiple Sequence Alignment Tool. Based on above findings, it is concluded that the optimized PCR conditions and the buffalo COX-2 gene specific primers can be useful in pursuing further gene expression studies to explore the function of COX-2 gene in buffalo.