Mirna first strand cdna synthesis kit

The total RNA was extracted using Biozol reagent. The miRNA first-strand cDNA synthesis kit and miRNA Real-Time PCR Assay kit (aidlab, Beijing) were used to quantify the miRNA transcripts in our study following the manufacturer’s instructions. Each reaction sample was run in triplicate. The expression of U6 (F-CTCGCTTCGGCAGCACA, R-AACGCTTCACGAATTTGCGT) small nucleolar RNA was used as a control. All experiments were carried out in triplicate. The data were analyzed according to the comparative CT method (2^−ΔΔCt).

We perform Real-time quantitative PCR analysis for randomly selected 5 known differentially expressed miRNAs and 3 novel miRNAs. First-strand cDNA was synthesized using miRNA first-strand cDNA synthesis kit (Aidlab Biotechnology Co. Ltd., Beijing, ChinaqRT-PCR was carried out using the TransStart^® Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). The qRT-PCR was performed using mRNA-specific primers and a universal miRNA reverse primer 5′-TCTAGAGGCCGAGGCGGCCGACATGT-3′. The primer sequences are listed in the Table 1. U6 gene and β-actin gene were used as endogenous internal controls for normalization. We collected cells before transfection as a control group. The cells in this control group were not treated with mimic, inhibitor, mimic NC, inhibitor NC. When studying the expression of gga-miR-454 during the growth of primary myoblasts, we counted the gga-miR-454 expression collected at 24 h as 1, and compared it with other time points to calculate the difference. The 2^–ΔΔCT method was used to determine the relative miRNA and mRNA abundance (Livak and Schmittgen, 2001 (link)).

Total RNA was extracted. The miRNA first-strand cDNA synthesis kit and miRNA Real-Time PCR Assay kit (Aidlab, Beijing) were applied to quantify the miRNA transcripts. U6 small nucleolar RNA was used as reference. Each reaction sample was run in triplicate. The relative expression level of miRNA was calculated using the comparative CT method (2^−ΔΔCt).

To confirm the results of high-throughput sequencing, six miRNAs, including three novel miRNAs and three conserved miRNAs, were randomly selected for RT-qPCR for each comparison. The CTAB method was used to extract RNAs from each sample [72 ,73 (link)]. Mature miRNA reverse transcription was performed by using the miRNA First-Strand cDNA Synthesis Kit (Aidlab Biotechnologies, Beijing, China). A miRNA Real-Time PCR assay kit (Aidlab Biotechnologies, Beijing, China) was used for the RT-qPCR analysis. Briefly, 20 μL reaction volume with 0.5 μL cDNA, 0.4 μM of each primer, 10.0 μL of 2× miRNA qPCR Mix, and 8.7 μL ddH₂O was mixed in 96-well plates with a StepOne Plus PCR System (Applied Biosystems, Foster City, CA, USA) under the following default cycling conditions (40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s). All of the reactions were performed in triplicate. peu-5.8s rRNA was used as an internal control for miRNA [81 (link)]. To further confirm the relationships between miRNAs and their target genes, UBQ was used as an internal control for target genes, and the 2^-ΔΔCT method was adopted to calculate the relative expression of RT-qPCR [82 (link)]. All the primers used in this study are listed in Table S1.