Mircute plus mirna qpcr kit

Total RNA was separated with Trizol reagent and reverse‐transcribed into cDNA using the miRcute Plus miRNA First‐Strand cDNA kit (Tiangen) for miRNA or FastKing RT kit (Tiangen) for mRNA. miRNA was quantified using the miRcute Plus miRNA qPCR kit (Tiangen), and mRNA was quantified using the SuperReal PreMix Plus kit (Tiangen). Gene expression was normalized to U6 or GAPDH and calculated by the 2^−ΔΔCt method. See Table S1 for polymerase chain reaction (PCR) sequences.

Total miRNAs were prepared from cultured cells using TRIzol reagent (Thermo) and phenol–chloroform extraction. Specific miRNA primers formiR-30a was as follows: 5′-ACACTCCAGCTGGGTGTAAACATCCTCGAC-3′ (forward) and 5′-CAGTGCGTGTCGTGGAGT-3′ (reverse). The miRcute miRNA cDNA kit (Tiangen Biotech Co., Ltd) was used to reverse transcribe cDNA, and the miRcute Plus miRNA qPCR Kit (Tiangen Biotech Co., Ltd) was used to detect RT products. The thermal cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 20 s. The relative expression was calculated with the 2^−ΔΔCt method.

For the RT–qPCR analysis, reverse transcription was performed using a PrimeScript™ RT reagent kit (TaKaRa) and miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, Beijing, China) for mRNA and miRNA according to the manufacturers’ instructions and previous study [55 (link)], respectively. RT–qPCR was performed by a RocheLight Cycler®480 II system (Roche Applied Science, Mannheim, Germany) with SYBR Green qPCR Mix Kit (TaKaRa, Dalian, China) and miRcute Plus miRNA qPCR Kit (TIANGEN, Beijing, China) for mRNAs and miRNAs, respectively. RT–qPCR analysis of mRNA and miRNA expression was conducted by the following procedure: for mRNA, initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s; for miRNAs, initial denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 94 °C for 20 s and annealing at 60 °C for 34 s. The data were analyzed with the 2^-∆∆Ct method. The goat PRL19 gene and U6 were used as reference genes for the normalization of the target gene data. The sequences of the RT–qPCR primers are listed in Supplementary Table S6.

First, miRNA was isolated using a miRcute miRNA isolation kit (Tiangen, Beijing, China). First‐strand cDNA was then generated using a miRcute miRNA cDNA synthesis kit (Tiangen) and detected using a miRcute Plus miRNA qPCR kit according to the manufacturer's protocols. Forward primers of miR‐199a‐5p (cat.CD201‐0272), miR‐501‐5p (cat.CD201‐0641), and U6 snRNA (cat.CD201‐0145) were purchased from TIANGEN BIOTECH CO., LTD. The relative miRNA expression was normalized to U6 expression.
Total RNA was isolated from HCC cell lines or tissue samples using Invitrogen TRIzol. A PARIS kit (Life Technologies, Carlsbad, CA, USA) was used to separate and purify cytoplasmic and nuclear RNA. A NanoDrop 2000 spectrophotometer was used to determine the extracted RNA concentration. For reverse‐transcription PCR, cDNA was synthesized using a cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA). Then, qRT‐PCR was conducted on an ABI PRISM™7900 HT sequence detection system (Applied Biosystems) using SYBR Green I (Takara, Japan). The primers for lincRNAs and other genes are summarized in Table S6. The relative expression of lincRNAs and mRNAs was normalized to 18s rRNA expression and GAPDH expression, respectively. The RNA expression was quantified using the 2^−ΔCt (ΔCt = Ct (Target) – Ct (Reference)) method.