Total RNA were isolated from the five taproot samples (10, 15, 20, 40 and 50 DAS, respectively) using Trizol reagent (Invitrogen, USA) and then treated with PrimeScript® RT reagent Kit (Takara, Dalian, China) to reverse transcribe into cDNA. MicroRNA was extracted from five radish taproot samples using RNAiso for small RNA kit (Takara, Dalian, China) and reverse transcribed into cDNA using a One Step PrimeScript® miRNA cDNA Synthesis Kit (Takara, Dalian, China). The cDNA was quantified by an iCycler IQ real-time PCR detection system (BIO-RAD) using a 20 μl reaction mixture, which consisted of 2 μl of diluted cDNA, 0.2 μM forward and reverse primer, and 10 μl of 2× SYBR Green PCR Master Mix (Takara, Dalian, China). The amplification reaction for miRNAs and their targets was performed, respectively, according to the previous reports [24 (link),25 (link),31 (link)]. The equation ratio 2−ΔΔCτ was applied to calculate the relative expression level of miRNAs and targets using 5.8S rRNA and Actin gene as the reference gene, respectively. The primers for real-time RT-qPCR were designed using Beacon Designer 7.0 software (Additional file 1 A and B). In addition, the statistical analysis with SAS Version 9.0 software (SAS Institute, Cary, North Carolina, USA) was performed using Duncan’s multiple range test at the P < 0.05 level of significance.
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