Miscript plant rt kit

Gene expression analysis (miRNA and mRNA) was performed via reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). First, template cDNA was synthesized from 500 ng of total RNA using a PrimeScript RT reagent kit with gDNA eraser (Takara bio, Kusatsu, Japan), miScript II RT kit (Qiagen), and miScript Plant RT kit (Qiagen) for mRNA, premature miRNA, and mature miRNA amplification, respectively, according to the manufacturer’s instructions. Target genes were amplified using SYBR Premix Ex Taq II (Takara Bio) for mRNA and miScript SYBR Green PCR kit (Qiagen) for miRNA. For mature miRNA amplification, we designed specific mature miRNA sequences for the forward primer and used the universal primer provided in the miScript SYBR Green PCR kit as the reverse primer. Primer sequences used in this study are listed in Table S1 (in supplementary data). RT-qPCR was performed using a LightCycler® 96 Instrument (Roche, Bazel, Switzerland). The relative gene expression level was calculated using the comparative Ct method.^{30 (link)}OsGAPDH and snoR31 were used as reference genes for mRNA and miRNAs, respectively. The relative expression level of premature miRNA calculated using snoR31 presented no significant difference compared with that calculated using U6, a previously known reference gene for miRNA analysis (Figure S1 in supplementary data).^{16 (link)}

Total RNA was extracted using a Pure Cell/Bacteria kit (Tiangen, Beijing, China). The miScript Plant RT Kit (Qiagen, Hilden, Germany) designed for small RNAs 3′end with 2′-O-Me modification was used for small RNA reverse transcription. The miScript SYBR Green PCR Kit (Qiagen) was used for real-time PCR. The specific primer sequence for piR-823 amplification was 5′-AGCGTTGGTGGTATAGTGGT-3′. The piR-823 normalization was set up by Hs_SNORD61_11 miScript Primer Assay (Qiagen). The FastQuant RT Kit (Tiangen) was applied for total RNA reverse transcription into cDNA. The quantity of cDNA was analyzed by SuperReal PreMix Plus (Tiangen). GAPDH was used as an internal control. The specific primers of α-SMA, col1a1, EIF3B, TGF-β1, and GAPDH are shown in Table 2, and relative expression was calculated using the 2^−ΔΔCt method.