Platinum sybr green based qpcr

We performed qPCR to explore the validation and stress expression patterns of identified miRNAs. The expression of selected miRNAs was assayed in rapeseed by Platinum SYBR Green based qPCR (Invitrogen, 11733–038) with the High Specificity miRNA QuantiMir RT Kit (RA610A-1, System Biosciences) on Step One^™ Real-Time PCR System (Applied Biosystems). A total of 10 conserved miRNAs, 4 novel miRNAs and 3 corresponding target genes were validated by qPCR technology. These miRNAs and the internal control genes (5.8S rRNA) are available in Table A in S1 File. The qPCR reactions were performed as follow: 94°C for 30 s, and then 40 cycles at 94°C for 10 s, 58°C for 30 s. All the gene expression data were obtained from three individual biological/technical replicates and processed according to previous analyses [27 (link),28 ].

The expression of five selected miRNAs were assayed in two lines of rice by Platinum SYBR Green-based q-PCR (Invitrogen, 11733–038) with the High Specificity miRNA QuantiMir RT Kit (RA610A-1, System Biosciences) on ABI 7900. Primers for the five miRNAs and internal control gene U6 snRNA are listed in Table S8.

To confirm the predicted results, qRT-PCR for miRNAs detection was performed to examine their expression. Fourteen miRNAs were randomly selected for the qRT-PCR assays in the samples of C. gynandra and C. hassleriana by Platinum SYBR Green-based qPCR (Invitrogen, USA) with the High-Specificity miRNA QuantiMir RT Kit (RA610A-1, System Biosciences) on the ViiA™ 7 Dx platform (ABI, USA). Amplified primers for all miRNAs were designed according to Varkonyi-Gasic et al.53 . The miRNA-specific forward primers of the 14 selected miRNAs and the internal control U6 are listed in the supplementary table (Supplementary Table 5). The qRT-PCR procedure was as follows: 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 30 s, and a final step at 95 °C for 15 s, 60 °C 1 min and 95 °C for 15 s. After the qRT-PCR amplification, the melting curve and amplification curve were examined to evaluate the specific amplification. The relative expression levels of the miRNAs were analyzed by the 2^−ΔΔct method, and U6 was used as the internal control. All qRT-PCR reactions were assayed in triplicates.