Mircute mirna first strand cdna synthesis

Total RNA was extracted with TRIzol Reagent (Invitrogen). Reverse transcription of total RNA was performed using Prime-Script RT reagent Kit (TaKaRa, Dalian, China). Quantitative real time PCR to measure ATF5 expression was carried out with SYBR^® Green PCR Kit (Qiagen, Germany). Micro-RNA was isolated by miRcute miRNA isolation kit (Tiangen, Beijing, China). Reverse transcription of miRNA was performed using miRcute miRNA First-Strand cDNA Synthesis (Tiangen). Quantitative real time PCR to measure miRNA expression was carried out with miRcute miRNA qRT-PCR Detection Kit (Tiangen). Primers used were shown as following: ATF5 forward 5′-AAGTCGGCGGCTCTGAGGTA-3′ and reverse 5′-GGACTCTGCCCGTTCCTTCA-3′, human β-actin forward 5′-CCCAGCACAATGAAGATCAA-3′ and reverse 5′-GATCCACACGGAGTACTTG-3′, GAPDH forward 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse 5′-GAAGATGGTGATGGGATTTC-3′. In addition, primers of miR-141-3p and U6 were purchased from Tiangen. The mRNA expression of ATF5 was normalized with β-actin and GAPDH, miR-141 expression was normalized with U6. qRT-PCR was performed on iQ5 Real-Time PCR Detection system (Bio-Rad, USA), the results were analyzed by the 2^−ΔΔCt method.