Takara cdna reverse transcription kit

The RNA was extracted by TRIzol^® Reagent (Life Technologies, Carlsbad, CA, USA) and reverse transcribed to cDNA using Takara cDNA Reverse Transcription Kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. The qPCR was performed based on the resulting cDNA with specific primers in the Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Predesigned primer pairs for mouse and human PTP4A3, mouse and human β-Actin, were purchased from Sangon (Shanghai, China). The primers for each gene are listed in Table 1. According to the manufacturer’s instructions, each well required 20 μL reaction mixture, including the following components: 10µL of SYBR Premix Ex Taq II, 0.8µL of each forward and reverse primer, 0.4µL ROX Dye and 500 ng template cDNA. The qPCR procedure was designed as below: the denaturation stage was 95 °C for 5 s, followed by the amplification stage (95 °C for 15 s, 60 °C for 60 s and 95 °C for 15 s) with 40 cycles. The 2^−ΔΔCt method was used to analyze the gene expression. The expressions of target genes were normalized based on β-Actin. The results were adjusted according to the primer efficiencies previously calculated.

Total RNA was extracted from cells with the Genezol reagent (Genaid, Taiwan) and was submitted to reverse transcription using the TAKARA cDNA Reverse Transcription kit (Takara, Japan). Quantitative PCR was performed with the fluorescent dye SYBR Green methodology and a Rotor-Gene Q real-time PCR system. Primers for gene expression analysis were purchased from IDT (Singapore). These mean Cq values were used to normalize the steady-state target mRNA concentrations to those of the beta-actin by the 2(−ΔΔCq) method.