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

Characterization of COX-2 gene in endometrial epithelial cells of buffalo

Abstract

 Cyclooxygenase-2 (COX-2), also known as prostaglandin-endoperoxide H synthase-2 plays a vital role in various reproductive events including prostaglandin biosynthesis. 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. After initial denaturation at 98°C for 3 min, 30 cycles of amplification were carried out under the following conditions: denaturation at 98°C for 45 sec, annealing at 59°C for 30 sec, initial extension at 72°C for 90 sec and final extension at 72°C for 10 min. The 449 bp PCR product was checked on 1% agarose gel to verify the amplification of the target region. The optimum annealing temperature for the primers was found to be 59°C for specific amplification of 449 bp. 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.