Small RNAs are key molecules in RNA silencing pathways that exert the sequence-specific regulation of gene expression and chromatin modifications in many eukaryotes. In plants, endogenous small RNAs, including microRNAs (miRNAs), trans-acting short interfering RNAs (tasiRNAs), and heterochromatic siRNAs (hc-siRNAs), play an important role in switching or orchestrating biological processes during the development and at the onset of stress responses. These endogenous and exogenous small RNAs are mainly 20-24 nucleotides in length. In addition, viral genome-derived siRNAs of similar lengths are produced during viral infection, and they exhibit anti-viral defense activity in RNA silencing pathway.Here, we introduce a method to isolate and characterize small RNA molecules possibly applicable to a wide range of plant resources and tissues. After purification from total RNAs, small RNAs were subjected to Illumina sequencing analysis using compatible reagents kits. Following the sample preparation protocol, small RNAs are ligated first at the 3'- and then at the 5'-end to the respective RNA adapters followed by reverse transcription with a set of primers to produce cDNAs with Index sequences at ends. After PCR amplification, cDNAs are subjected (after gel purification) to RNA-seq analysis. This method could be applied to isolate small RNAs from different sources and characterize small RNA profiles to compare different sets of samples, e.g., wild-type and mutant plants, plants under different stress environments, and virus-infected plants because the starting RNA material is free of contaminated starch or similar material which would block further analysis.
Keywords: Cloning; Sequencing; Small RNA; Virus-derived siRNA; miRNA; siRNA.