Access reverse transcription polymerase chain reaction rt pcr system

Complementary DNA (cDNA) was produced from the isolated RNA using the Access Reverse transcription-polymerase chain reaction (RT-PCR) System (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. The PCR mixture contained one mM antisense and sense primers (Table 1).
First-strand cDNA was synthesized under the following conditions: 45 °C for 45 min (1 cycle) for reverse transcription, followed by 94 °C for 2 min (1 cycle) for avian myeloblastosis virus reverse transcriptase inactivation and RNA/cDNA/primer denaturation. The following conditions were used for the second-strand cDNA synthesis and PCR amplification: 94 °C for 30 s for denaturation; 60 °C for 1 min for annealing; 68 °C for 2 min for extension, 40 cycles; and 68 °C for 7 min for final extension, 1 cycle.

Total RNA was extracted from tiny slices of pancreatic tissues taken from animals in each subgroup using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription of RNA into complementary DNA (cDNA) was performed using the Access Reverse transcription-polymerase chain reaction (RT-PCR) system (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. Real-time PCR was performed in a PCR system using gene-specific forward and reverse primers, as specified in Table 1, to determine the expression of mRNAs in pancreatic tissues using Actb as a reference.

SV Total RNA Isolation System, Access reverse transcription polymerase chain reaction (RT-PCR) System, and T4 DNA ligase were purchased from Promega (Madison, WI, USA). The restriction enzymes of NcoI and XhoI were purchased from NEB (Ipswich, MA, USA). Competent cell NovaBlue, pTriEx-1.1 Hygro vector, and GeneJuice® transfection reagent were supplied by Novagen (Billerica, MA, USA). Nickel–nitrilotriacetic acid (Ni–NTA) His Bindresin was purchased from Qiagen (Hilden, Germany). HEK293 and horseradish peroxidase (HRP)-β-agonists were supplied by Beijing Kwinbon Biotechnology Co., Ltd (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY, USA). Anti-His monoclonal antibody and HRP-conjugated goat anti-mouse IgG were obtained from Beijing ComWin Biotech Co., Ltd (Beijing, China).
CBL, SAL, and ractopamine (RAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). HRP-β-agonists were gifts from Beijing Kwinbon Biotechnology Co., Ltd (Beijing, China). All chemicals were of analytical grade without any further purification.