Dreamtaq green pcr mix

Embryos were snap frozen in liquid nitrogen, total mRNA was extracted using the Direct-zol RNA MiniPrep kit (ZYMO research, Irvine, CA, USA), and first-strand complementary DNA (cDNA) was generated using the iSrcipt reverse transcription mix (Bio-Rad Hercules, CA, USA). Quantitative PCR products were generated using the DreamTaq Green PCR mix (ThermoFisher scientific, Waltham, MA, USA). The primers used for qPCR allowed amplification of ribeye a, ribeye b, GluR2/3, Cav1.4, Cav1.3, and 18S ribosomal RNA (rRNA) for normalization are listed in Table 2.

Dulbecco’s Modified Eagle Medium (DMEM), trypsin, and fetal bovine serum (FBS) were purchased from Welgene (Gyeongsan, Republic of Korea). Streptomycin–penicillin was purchased from Gibco (NY, USA). Calcium-free DMEM, superscript™ IV first-strand synthesis system, and Dream taq Green PCR Mix were purchased from Thermo Fisher Scientific (MA, USA). TRIzol reagent was purchased from Invitrogen (CA, USA). Sodium dodecyl sulfate (SDS), dithiothreitol (DTT), agarose, lipopolysaccharide (LPS), ethyl ether, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Griess reagent, NG-Methyl-l-arginine acetate salt (L-NMMA), and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma Aldrich (St. Louis, USA). Reagent set B, cytokine IL-6 and IL-8 ELISA sets used for immunoassay were purchased from BD Biosciences (NJ, USA). TI was acquired in a previous study [31 (link)].

RNA was extracted from cells using an RNeasy Mini kit (Qiagen, Maryland, USA) and its quantity and quality were estimated by NanoDrop measurement (Thermofisher, USA). 500 ng RNA was used for cDNA synthesis (Qiagen) and gene expression monitored by Real-time PCR with SYBR Green master mix using commercially available RT2 Profiler PCR Array and primers for genes of interest (Qiagen, USA). Amplicons were generated on the QuantStudio Flex 12K (Applied Biosystems, California, USA) and gene expression calculated using the comparative threshold cycle method (2^−ΔΔCt) after normalising with transcripts encoding HPRT1 and YY1.
For RT-PCR experiments, purified RNA was reverse transcribed into cDNA using SuperScript VILO cDNA synthesis Kit (Roche), according to the manufacturer’s instructions. DNA was amplified by cycles of denaturation (95 °C, 30 s) annealing (primer-specific temperature; 30 s) and extension (72 °C; 30 s) in DreamTaq Green PCR Mix (ThermoFisher) for 35–40 cycles. Final extension was for 5 min at 72 °C. Amplified PCR products were separated and visualised after electrophoresis in 1% agarose gels containing 0.005% GelRed. Vomeronasal receptor primer details are provided in Supplementary Table 1.