Cfx96 touch realtime machine

Total RNA was isolated using peqGOLD TriFast^TM (VWR) according to manufacturer's instructions. cDNA synthesis and RT-qPCR were performed as described (25 (link), 29 (link)). For assays that are located in introns or exon-intron junctions, total RNA was DNase-treated prior to cDNA synthesis. Controls without reverse transcriptase were included for all RT-qPCRs. Primers for pre-mRNA analyses are listed in Supplementary Table 1. Sequences of primers and probes for Ube2d2 (ID 3377) and Irf1 (ID 3848) mRNA analysis are available at the Real-Time Primer and Probe Database (http://www.rtprimerdb.org/). Primers for Irf7 (QT00245266) and Irf8 (QT00174195) mRNA analysis were purchased from Qiagen. qPCRs were done in duplicate on a Bio-Rad CFX96 Touch™ realtime machine.

Total RNA was extracted from the OS tissue and cultured cells using TRIzol reagent (Invitrogen, CA, USA) and cDNA synthesis kit (Toyobo, Kyoto, Japan) was used in generating cDNA according to the manufacturer’s protocol. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using SYBR Green PCR kit (Toyobo, Kyoto, Japan) with an CFX96 Touch real time machine (Bio-Rad, USA) according to manufacturers’ instructions. DNA amplification was programmed for an initial 95 °C for 60 s, followed by 40 cycles of 95 °C 15 s, 60 °C 15 s and 72 °C 45 s, and ended with 72 °C 5min. The relative expression level of target gene mRNA normalized to β-actin was determined by the 2-ΔΔCt method. All the primers used in this study were showed in Table 1.