Dntps 10 mm

Total RNA was extracted from cells using TRIzol^® reagent (cat. no. 3101-100; Shanghai Pufei Biotechnology Co., Ltd.). Total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase (cat. no. M1705; Promega Corporation). The Oligo dT was provided by Sangon Biotech Co., Ltd. (cat. no. B0205) and the dNTPs (10 mM) were provided by Promega Corporation (cat. no. U1240). After heat treatment of RNA/primer mixture at 70°C for 10 min and cooling immediately on ice for 10 min, the RT reaction was processed at 42°C for 1 h and 70°C for 10 min. Subsequently, qPCR was performed using ExTaq (Takara Bio, Inc.). The SYBR Master Mixture was provided by Takara Bio, Inc. (cat. no. DRR041B). The following primer were used for the qPCR: RRBP1 forward, 5′-GAGATGGCGAAAACTCACCAC-3′ and reverse, 5′-CTCGAAGGAGGACAGTCACAT-3′; CCR7 forward, 5′-TTCATCGGCGTCAAGTTCC-3′ and reverse, 5′-AAGGTGGTGGTGGTCTCG-3′; and GAPDH forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′. The following thermocycling conditions were used for the qPCR: Initial denaturation at 95°C for 30 sec; followed by 40 cycles of 95°C for 5 sec; and final extension at 60°C for 30 sec. mRNA expression levels were quantified using the 2^−ΔΔCq method (17 (link)) and normalized to the internal reference gene GAPDH.