Differential miRNA Expression in Photosynthesis

To identify and validate the differentially expressed miRNAs in the photosynthesis pathway, six miRNAs with high expression levels (at least 1000) in all libraries were selected from conserved families (miR171f, miR319, MiR156a, and miR159), and non-conserved (MIR305714, MIR46018, and MIR2836) and amplified using real-time qPCR to examine their expression. The forward miRNAs primers were designed based on the full miRNAs sequence, while the reverse primer was the universal reverse primer (5’GTGCAGGGTCCGAGGT3’). The stem-loop primer, used for miRNA cDNA synthesis, was designed according to Chen et al. (2005) (link). The 18S rRNA was taken as the reference gene. Similarly, the expression profiles of the respective miRNA target genes were assayed by real-time qPCR. In summary, a 10-µg aliquot of RNA was used for the RT reaction followed by the addition of oligo(dT) primer. The RT reaction was performed by MMLV reverse transcriptase (Toyobo, Osaka, Japan) according to the supplier’s manual. For real-time qPCR, ACTIN for bermudagrass was used as a housekeeping gene as the internal control. To calculate the relative expression, the 2^-ΔΔCt method was used and Student’s t-test was performed to compare differences in expression profile. The means were considered significantly different when P ≤ 0.05.