Abi 7300 real time pcr instrument

RNF5 expression was assessed using RT-qPCR. Total RNA was extracted from U251 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse-transcribed into cDNA using the Quant One-Step RT-PCR kit (Tiangen Biotech Co., Ltd.). qPCR was performed using FastStart Universal SYBR Green Mix (Roche Diagnostics) and an ABI 7300 real-time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers for RNF5 and β-actin were designed as follows: RNF5, forward 5′-GTACCCATACGATGTTCCAGATTACGC-3′, reverse 5′-CTGAGCAGCCAGAAAAAGAAAAAGATG-3′; and β-actin forward, 5′-CATGTACGTTGCTATCCAGGC-3′, and reverse, 5′-CGCTCGGTGAGGATCTTCATG-3′. Thermocycling conditions included pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec and extension at 72°C for 1 min for 35 cycles. Expression level of RFN5 was calculated using the 2^−ΔΔCq method (17 (link)).

The assay of qRT-PCR was performed as previously described (20 (link)). Briefly, total RNA was extracted from the cultured bEnd.3 cells (2×10⁵ cells/ml) using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. RNA was reverse transcribed using, and qPCR was performed with an ABI 9700 PCR Thermal Cycler and an SYBR-Green kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instructions. Briefly, qRT-PCR was performed using 10 µP 2X SYBR-Green PCR Master Mix (Toyobo Life Science, Osaka, Japan), with 5 µl of cDNA, 0.5 µl of primers and 4 µN of RNase-free ddH₂O contained in 20 µl of reaction mixture. The reaction was performed with one cycle of 95°C for 5 min and 40 cycles of 95°C for 15 sec, 65°C for 15 sec and 72°C for 35 sec in ABI 7300 real-time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.). When the reaction proceeded. Ct value was obtained, and results were analyzed using 2^−∆∆Cq calculation (20 (link)). β-actin was used to normalize the data. The primer sequences were: β-actin forward, 5′-CATTGCTGACAGGATGCAGA-3′ and reverse, 5′-CTGCTGGAAGGTGGACAGTGA-3′; PPARγ forward, 5′-GGAAGACCACTCGCATTCCTT-3′ and reverse, 5′-GTAATCAGCAACCATTGGGTCA-3′; BIRC5 forward, 5′-GAGGCTGGCTTCATCCACTG-3′ and reverse, 5′-ATGCTCCTCTATCGGGTTGTC-3′. β-actin was used as an internal control.