Qpcr detection kit

One-cell stage zebrafish embryos were injected with a solution consisting of 30 ng/μl CMV-Gal4-VP16 plasmid and 30 ng/μl pUAS-mCherry/pUAS-mCherry-mircoRNA/pUAS-mCherry-miR-shRNA. To detect miRNAs level, 3 days post fertilization (dpf) zebrafish larvae with relatively high mosaic red fluorescence were selected for total RNAs isolation by miRNA Isolation Kit (Tiangen), according to the manufacturer's protocols. Each sample was reverse-transcribed into cDNA by miRNA First-Strand cDNA Synthesis Kit (Tiangen) and was subjected to qRT-PCR analysis with qPCR Detection Kit (Tiangen). To detect mRNAs levels, 10 hpf zebrafish embryos expressing red fluorescence were selected to isolate total RNAs with the same kit mentioned above. Each sample was reverse-transcribed into cDNA with HiScriptII Q RT SuperMix (Vazyme) and was subjected to qRT-PCR analysis with AceQ qPCR SYBR Master Mix (Vazyme). Each experiment was carried out with three biological and experimental replicate. Results were shown as mean fold changes ±s.e.m. qRT–PCR primers were shown in Table S1.

Total RNA was extracted using TRIZOL Reagent (#15596-026, Invitrogen) according to the manufacture's protocol. For the detection of mature miRNA, miRNA was tailed and reverse transcribed using miRcute miRNA First-Strand cDNA Synthesis Kit (# KR201, TIANGEN), followed by qRT-PCR with SYBR Green chemistry using miRcute miRNA qPCR detection kit (#FP401, TIANGEN). Real-time PCR was performed in triplicate in a 384-well optical plate on the ABI ViiA™7 Sequence Detection System. The reactions were incubated at 94°C for 2 min, followed by 40 cycles of 94°C for 20 sec and 60°C for 34 sec. Analysis of relative miRNA expression data was performed using the 2^-ΔΔCt method [43 (link)] with the U6 small nuclear RNA as an endogenous control. Results were expressed as the amount of miR-324-5p normalized to U6 and relative to a calibrator (one LNM (-) PTC sample). The universal reversal primer was contained in the qPCR detection kit (#FP401, TIANGEN). The specific forward primers of miR-324-5p and U6 were purchased from Guangzhou RiboBio Co., Ltd. (Table 7).

All the synthetic miRNA mimics and inhibitors as well as scrambled miRNAs were purchased from Invitrogen. For the miRNA isolation and qPCR analysis, total RNA was extracted from the cells using the mirVana miRNA Isolation Kit. The miRcute miRNA First-strand cDNA Synthesis Kit (Tiangen, Beijing, China) and qPCR Detection Kit (Tiangen) were used to measure the expression levels of miR-25, -30b, -4458 and -210 before and after drug treatment; human U6 snRNA served as an internal control. For mRNA quantification, total RNA was isolated with TRIzol reagent, and reverse transcription was performed using a First-strand cDNA Synthesis Kit (Roche, Mannheim, Germany) according to the manufacturer's instructions. Real-time PCR was performed using a SYBR Green kit (Roche) on the Roche LightCycler 480 platform. The primers for miRNAs and mRNAs are described in Supplementary Table 1. The Ct value was measured during the exponential amplification phase. The relative expression level (defined as the fold change) of each target gene was determined using the 2^−ΔΔCt method and was normalized to the fold change.