Specific pcr primers

RNA was extracted from tissues or cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) following the manufacturer’s protocol. Total RNA (2 µg) was reversed transcribed into cDNA using the TaqMan microRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed in a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd., Dalian, China) or TaqMan probes (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions. The thermo-cycling conditions were as follows: 95°C for 5 min followed by 45 cycles at 95°C for 10 sec and 55°C for 30 sec, then a melting curve analysis every 0.2°C from 55°C to 95°C for 2 min was obtained. Expressions were normalized to the levels of the internal reference gene GAPDH. Specific PCR primers were synthesized by Invitrogen and the sequences were as follows: miR-214 forward, 5′-TAT ACA TCA AAC AGC AGG CAC A-3′ and reverse, 5′-CAT TCG ATC TTC TCC ACA GTC TC-3′; PSMD10 forward, 5′-CGA GCT CTG GAA GGT TAA ACA GCT TGG A-3′ and reverse, 5′-GCT CTA GAA GAC TCA CAA CAG CCA CAG AA-3′; GAPDH forward, 5′-AAG AAG GTG GTG AAG CAG GC-3′ and reverse, 5′-TCC ACC ACC CTG TTG CTG TA-3′. The expression levels of miR-214 and PSMD10 were assessed using the 2^−∆∆Cq method (22 (link)).

Total RNA was extracted from tissue or cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. RNA was reversed-transcribed into cDNA using PrimeScript Reverse Transcription kit (Takara Bio, Inc., Otsu, Japan). The qRT-PCR reactions were performed using SYBR Premix Ex Taq (Takara Bio, Inc.) on a 7500 ABI system (Applied Biosystems; Thermo Fisher Scientific, Inc). U6-siRNA was used for normalization. Specific PCR primers were synthesized at Invitrogen; Thermo Fisher Scientific, Inc. The primer sequences used were as follows: miR-125b, 5′-TCCCTGAGACCTAACTTGTG-3′ (forward); U6, 5′-ACGCAAATTCGTGAAGCGTT-3′ (forward), a universal primer (reverse); BMF, 5′-CCCATAAGCCAGGAAGACAA-3′ (forward), and 5′-CTGAAGCTTTCTGGCGATTCT-3′ (reverse).

Total RNA was extracted from cultured cells using Trizol reagent (Invitrogen, USA) and reverse-transcribed to cDNA using a PrimeScript RT Reagent Kit (TaKaRa, China) following the manufacturer’s protocol. qRT-PCR was performed to amplify the cDNA template using the SYBR Green PCR kit (TaKaRa, China). The levels of HULC and miR-218 were normalized to those of U6. The mRNA level of TPD52 was normalized to GAPDH. Specific PCR primers were synthesized at Invitrogen, USA. The relative expression was calculated using the 2^−ΔΔCT method.