Fluorescence detection system

Reverse transcription reactions were carried out using miRNA First-Strand cDNA Synthesis SuperMix (TransScript) according to the manufacturer’s instructions. A SYBR PrimeScript miRNA RT-PCR Kit was used to perform qRT-PCR in a fluorescence detection system (TianGen Biotech, Beijing, China) following the manufacturer’s instructions. All reactions were performed in triplicate for each sample. The 2^-∆∆CT method was then used to calculate the relative expression levels of the miRNAs [74 (link)]. Target genes of the miRNAs that were differentially expressed during grain development in the HN and LN treatments were selected to validate their expression patterns in developing wheat grain via qRT-PCR. Fisher’s least significant difference test (LSD) was subsequently used to distinguish differences in relative expression levels between different development stages using SPSS (Statistical Program for Social Science) software; P < 0.05 was considered statistically significant. All primer sequences used and the target gene identification results are given in Table S7 (Additional file 8).

The qPCR reactions were performed with a SYBR PrimeScript miRNA RT-PCR Kit on a Fluorescence detection system (TianGen Biotech, Beijing, China). Each 20 μl reaction contained 1 μl cDNA template (∼100 ng), 1 μl 10 μM PCR forward primer, 1 μl 10 μM Uni-miR qPCR primer, 10 μl 2× SYBR premix EX TaqII, and 7 μl ddH₂O. The amplification reactions were performed by first incubating at 95°C for 5 min, followed by 45 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 45 s. Following amplification, a threshold was set and the threshold cycle (CT) was recorded automatically. All reactions were performed in triplicate for each sample. The relative expression levels of the miRNAs were calculated using the 2^-ΔΔCT method (Livak and Schmittgen, 2001 (link)) and the data were normalized to the CT values for the Actin gene. The primer sequences for 16 differentially expressed miRNAs are given in Supplementary Table S13-1.