Dntps

Total RNA was extracted from cells using TRNzol reagent (TIANGEN, China) according to the manufacturer's protocol. First-strand cDNA was synthesized in a 20 μl of reaction volume using a random primer (TAKARA, Japan) and 1 μl reverse transcription enzyme M-MLV-RT (Promega, USA). PCR was performed using each specific primer set in a total volume of 20 μl containing 10 pmol of each primer, 4 μmol dNTPs (Solarbio, China), 1 unit of Taq DNA polymerase (TIANGEN, China), and 1 × PCR buffer. Primer sequences are summarized in Supplementary Table S1. PCR cycle parameters were 15 s at 94 °C, 15 s at 55 °C, and 20 s at 72 °C for 16–25 cycles (BiP, 22; CHOP, 25; XBP1, 25; 28S, 16) followed by 72 °C for 5 min. Aliquots (5 μl) of each reaction mixture were electrophoresed on 4.8% polyacrylamide gels.

Total RNA was extracted from BC cells with TriPure Isolation Reagent (BioTeke Corporation) and the concentration was determined using a NanoDrop 2000 UV spectrophotometer (Thermo Fisher Scientific, Inc.). Next, cDNA was obtained via reverse transcription using Super M-MLV Reverse Transcriptase (BioTeke Corporation), dNTPs (Beijing Solarbio Science & Technology Co., Ltd.) and RNase inhibitor (BioTeke Corporation). Finally, using the cDNA template, primers, SYBR Green (Millipore Sigma) and 2X Power Taq PCR MasterMix (BioTeke Corporation), qPCR was performed and the relative mRNA expression level was calculated using the 2^−ΔΔCq method (21 (link)). The thermocycling conditions for qPCR were as follows: For miRNA, 94°C for 4 min, followed by 40 cycles of 94°C for 15 sec, 60°C for 20 sec and 72°C for 15 sec; and for mRNA, 94°C for 5 min, followed by 40 cycles of 94°C for 15 sec, 60°C for 25 sec and 72°C for 30 sec. miRNA and mRNA expression levels were normalized to ribosomal 5S RNA and β-actin, respectively. The primers used for qPCR are presented in Table II.