miRNA detection by stem-loop RT-PCR and validation by quantitative real-time PCR

Total RNA was extracted from pooled ovules of fsv1 and Gui 99 at each stage using TRIzol (Invitrogen). Then, the stem-loop RT-PCR was used to detect miRNA expression according to Varkonyi-Gasic et al. (2007). The DNase I–treated total RNA was reverse-transcribed by miRNA-specific stem-loop primers. The reverse transcription was performed at 16° for 30 min, 30° for 30 sec (60 cycles), 42° for 30 sec, 50° for 1 sec, and then 70° for 5 min. The rules in designing the stem-loop primers were based on Chen et al. (2005). The quantitative real-time PCR was carried out using the ABI Step One Plus Real-Time PCR System (Applied Biosystems) with the Thunderbird SYBR qPCR mix (Toyobo, Japan). The quantitative real-time PCR amplification reactions were performed under the following cycling parameters: 95° for 30 sec, 95° for 10 sec, 56° for 30 sec (40 cycles), and 72° for 15 sec. U6 was used as internal control for the miRNAs. Three biological replicates and two technological replicates were implemented for each sample and the results were represented as the mean ± SD of these replicates. In this study, four novel miRNAs were randomly selected and then detected by gel electrophoresis after stem-loop RT-PCR. The primers used in stem-loop RT-PCR and quantitative real-time PCR are all listed in Supplemental Material, Table S1.