M mlv rtase cdna synthesis kit

To test the relatively expression ratio of the egfp gene, total RNA was extracted from T-DNA integrated transformants by means of RNaiso plus (Takara) according to the manufacturer’s protocol. The first strand of cDNA was prepared with 5 μg of total RNA using the M-MLV RTase cDNA synthesis kit (Takara). Quantitative real-time PCR (qRT-PCR) was performed to test the relatively expression ratio of the egfp gene with the ABI ViiA7 Real-Time PCR System (Applied Biosystems, Foster City, CA). β-tubulin gene was as the endogenous control and the qRT-PCR conditions were 95 °C for 1 min, followed by 40 cycles of 95 °C for 30 s, and 60 °C for 30 s. The primer sequences for egfp gene and β-tubulin gene are listed in Table 1. Each reaction was replicated three times to estimate error and the gene expression was normalized by the 2^−ΔΔCt analysis.

Total RNA (including miRNA) was extracted using the miRNeasy Mini kit (Qiagen). Residual genomic DNA was eliminated with RNase‐free DNase I (Promega). To analyze miR530 expression, miRNA first‐strand cDNA was synthesized with the miRcute miRNA First‐Strand cDNA Synthesis kit (Tiangen, Beijing, China). The expression of miR530 was then quantified by qRT‐PCR with the SYBR PrimeScript™ miRNA RT‐PCR kit (Tiangen) with 5.8S rRNA as the internal control. To analyze the expression of the OsmiR530 precursor, the target mimic and OsPL3, c. 2 μg total RNA for each sample was reverse‐transcribed with the M‐MLV RTase cDNA synthesis kit (TaKaRa, Dalian, China). The resulting cDNA was used as the template for a qRT‐PCR assay, which was completed with the SYBR Green PCR master mix (TaKaRa). The OsEF‐1α gene was used as the internal control. The qRT‐PCR analysis involved three biological replicates for each miRNA and gene. The relative expression ratios of OsmiR530, its precursor OsMIR530 and OsPL3 were calculated according to the delta‐delta threshold cycle relative quantification method. Details regarding the qRT‐PCR primers are provided in Table S1.