2.2 ǀ RNA isolation, cDNA synthesis, amplification, sequencing and characterization of cpOAS1 gene
For amplification and mRNA expression of the cpOAS1 gene, total RNA isolation and cDNA synthesis were carried out. Total RNA was isolated from 50 mg of collected endometrial tissue using Tri reagent (Sigma, USA) as per the manufacturer’s instructions. The quality and integrity of RNA were checked by agarose gel electrophoresis and the quantity of total RNA was measured by a Qubit 3 Fluorometer (Invitrogen, USA). Further, cDNA was synthesized using total RNA (2μg) by the Revert Aid first-strand cDNA reverse transcription system (Fermentas, USA). After the termination of cDNA, each cDNA preparation was diluted four times with nuclease-free water and stored at -20°C till further use.
A pair of degenerate primers was designed based on a predicted sequence (accession number XM_005691488) and used for amplification of the cpOAS1 gene (Table 1). PCR amplification was carried out in a total volume of 25 μl of reaction mixture containing 20 pM of each primer and 5μl diluted cDNA by using a 2x master mixture (Sigma, USA) in a thermal cycler (Bio-Rad, USA). The PCR protocol included an initial denaturation at 94°C for 4 min followed by 34 cycles of denaturation (94°C for 30 s), annealing (56°C for 30 s) and extension (72°C for 1 min) followed by one cycle of final extension (72°C for 10 min). The PCR product was resolved by agarose gel (0.7%) electrophoresis and visualized by SYBR safe gel staining (Invitrogen, USA) under the Gel-Doc system (Molecular image XR+, Bio-Rad, USA). The amplified product was purified by using the HiPurATMPCR product purification kit (Himedia Laboratories, India) as per the manufacturer’s instructions. Purified PCR product was sent for forward and reverse sequencing using an ABI PRISM automatic sequencer under standard cycle conditions of Sanger’s dideoxy chain termination method. These sequences were subjected to BLAST analysis (www.ncbi.nlm.nih.gov/BLAST). The nucleotide and the deduced amino acid sequences were aligned with others sequences of various species. Pairwise identity and phylogenetic evolutionary analysis were conducted using the DNA Star tool.