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.