Mirna qpcr kit

After concentration and purity determination of RNA samples, they were subjected to reverse transcription to synthesize cDNA samples, with U6 and GAPDH as internal reference, respectively. SYBR Green (Applied Biosystems, USA) premix, template, forward/reverse primer and DEPC (Beyotime Biotechnology, China) were used to prepare the PCR reaction solution which was subjected to PCR amplification on a real‐time PCR amplifier. miRNA RT Kit from Shanghai TIANGEN was used for miRNA's PCR reaction. After miRNA was reverse transcribed into cDNA, PCR reaction and quantitative analysis of miRNA were performed as per the instructions mentioned in the MiRNA qPCR kit (TIANGEN, Shanghai). The primer sequences are shown below:
miR‐1281 (F: 5′ ACACTCCAGCTGGGTCGCCTCCTCC 3′, R: 5′ CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGGAGAGG 3′); U6 (F: CTCGCTTCGGCAGCAGCACATATA, R: AAATATGGAACGCTTCACGA); GIHCG (F: 5′CTTTCAAGAAGTTTGGCTGTC 3′, R: 5′ GCTCATTCAACGGATAAGTC 3′), GAPDH (F: 5′GAAGAGAGAGACCCTCACGCTG3′, R: 5′ACTGTGAGGAGGGGAGATTCAGT3′); TLE1 (NCBI Gene ID: 7088) (F: 5′GAGTCCCTGGACCGGATTAAA3′, R: 5′ AATACATCACATAGTGCCTCTGC 3′).

After the concentration and purity of RNA samples were tested, cDNA samples were synthesized by reverse transcription with U6 and GAPDH as the internal reference, respectively. SYBR Green (Applied Biosystems, USA) premixed solution, templates, forward (F)/reverse (R) primers, and DEPC (Beyotime Biotechnology, China) were prepared into PCR solution, which was placed on a RT‐PCR instrument for PCR amplification. MiRNAs were reversely transcribed into cDNAs by miRNA RT Kit [Tiangen Biotech (Shanghai) Co., Ltd.], and PCR and quantitative analysis of miRNAs were carried out according to miRNA qPCR kit instructions [Tiangen Biotech (Shanghai) Co., Ltd.]. 2^‐△△ct method was applied to calculate the relative expression levels and the control genes were U6 and GAPDH. Primer sequences are listed below:
miR‐656‐3p (F: 5'ACACTCCAGCTGGGAATATTATACAGTCA 3',.
R: 5'CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGAGGUUG 3');
U6 (F: CTCGCTTCGGCAGCAGCACATATA，R: AAATATGGAACGCTTCACGA);
NORAD (F: 5'TGATAGGATACATCTTGGACATGGA3'),
R: 5'AACCTAATGAACAAGTCCTGACATACA3');
GAPDH (F: 5'GAAGAGAGAGACCCTCACGCTG3', R: 5'ACTGTGAGGAGGGGAGATTCAGT3');
AKT1 (F: 5'AGCGACGTGGCTATTGTGAAG3', R: 5'GCCATCATTCTTGAGGAGGAAGT3').

On the night before spawning, the paired adult zebrafish are placed separately on both sides of the breeding tank, and the divider is opened at 8:30 a.m. on the spawning day to start mating. One-cell stage embryos were placed on a fluted agarose plate containing a small amount of EM solution. Approximately 1 nL of the mix solution or mRNA was injected per embryo.
Total miRNA was isolated from the 4 dpf (day post fertilization) larvae which expressed red fluorescence using the miRNA Isolation Kit (Tiangen, DP501, China), according to the manufacturer’s protocols. RNA concentration was measured by Nanodrop (Thermo Scientific, Waltham, USA). 1 μg miRNA was reversely transcribed into cDNA with miRNA First-Strand cDNA Kit (Tiangen, KR211, China) and cDNA was quantified by qPCR system (Light-Cycler 96, Roche) with miRNA qPCR Kit (Tiangen, FP411, China).To detect RNA levels, 4 dpf larvae with red fluorescence were selected to isolate total RNA using TRIzol (TARAKA, China), and the RNA was reverse-transcribed into cDNA with HiScript II qRT SuperMix II (Vazyme, China). The mRNA expression levels were quantified by the qPCR system using AceQ qPCR SYBR Master Mix (Vazyme, China). Twenty-five larvae were collected for each experimental condition. Each experiment was repeated three times in biology and technology. The qPCR primers used are listed in the Supplementary Table S1.