One step primerscript mirna cdna synthesis kit

The equal amounts of radish leaves from three independent biological replicates at vegetative and reproductive stages were pooled and used for a radish leaf transcriptome library construction. Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. The transcriptome library was prepared from the mixed leaves using an Illumina TruSeq RNA Sample PrepKit following the manufacturer’s instructions. Two small RNA (sRNA) libraries from leaves at vegetative stage (NAU-VS) and reproductive stage (NAU-RS) were constructed following previously reported procedures, respectively19 20 (link). Briefly, the sRNAs sized at 18–30 nt were separated and gel-purified on a 15% polyacrylamide denaturing gel from the total RNAs of the two samples. Then the isolated sRNAs were ligated to 5′- and 3′-RNA adaptors by T4 RNA ligase (TaKaRa) and transcribed to single-stranded cDNA using One Step PrimerScript miRNA cDNA Synthesis Kit (TaKaRa). Both small RNAs and transcriptome were sequenced using Illumina HiSeq™ 2000 at Beijing Genomics Institute (BGI), Shenzhen, China.

Quantitative reverse transcription-PCR (qRT–PCR) was employed to evaluate the validity of small RNA sequencing and also to analyze the expression patterns of miRNAs and their targets during different stages. miRNAs and total RNAs were extracted from samples and reverse-transcribed to cDNA using the One Step Primer Script® miRNA cDNA Synthesis Kit (Takara Bio Inc., Dalian, China) and SuperScript® III Reverse Transcriptase (Invitrogen, USA) following the manufacturer's instructions, respectively. All reactions were performed on a BioRad iQ5 sequence detection system (BIO-RAD) and carried out in a total volume of 20 μl including 0.2 μM primer pairs, 2 μl diluted cDNA, and 10 μl 2 × SYBR Green PCR Master Mix (TaKaRa). The PCR amplification reaction was performed following the previous reports (Zhai et al., 2014 (link)). The 5.8S ribosomal RNA (rRNA) was used as the reference gene for normalization. All reactions were done in triplicate, the 2^−ΔΔC_T method was used to calculate the relative expression data (Livak and Schmittgen, 2001 (link)). The statistical analysis was performed using SPSS 20 software (SPSS Inc., USA) with Duncan's multiple range test at the 5% level of significance. The primers for qRT–PCR were showed in Table S1.

Total RNA was extracted from cells with TRIzol reagents (Invitrogen) according to the manufacturer's protocol. Measure the absorbance at 260 nm to determine the concentration of total RNA. 1 µg of RNA of each sample was reversely transcribed to cDNA by One Step Primerscript miRNA cDNA Synthesis kit (Takara, Dalian, China) and PrimeScript RT reagent Kit (Takara, Dalian, China). QRT-PCR was carried out by the SYBR green Premix Ex Taq II (Takara) with Applied Biosystems StepOne Plus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The expression of U6 was used as endogenous control for analysis of miRNAs expression and GAPDH was used as endogenous control for analysis of other mRNA expression. The primers were synthesized by Sangon Biotech, Shanghai, China. Relative quantification was analyzed with the comparative △CT values.