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.
Cloning Small RNA siRNA miRNA Virus-derived siRNA Sequencing
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
We thank Drs. Minami Matsui and Yukio Kurihara at RIKEN CSRS for kind advice on sequencing. We also thank Drs. Takayuki Kohchi at Kyoto University and John Bowman at Monash University for sharing genome data information about M. polymorpha.
Axtell MJ (2013) Classification and comparison of small RNAs from plants. Ann Rev Plant Biol 64:137–159CrossRefGoogle Scholar
Tagami Y, Inaba N, Watanabe Y (2010) Cloning new small RNA sequences. In: Kovalchuk I, Zemp FJ (eds) Plant epigenetics, Methods in molecular biology. Springer, New York, NY, pp 123–138CrossRefGoogle Scholar
Toedling J, Ciaudo C, Voinnet O, Heard E, Barillot E (2010) girafe - an R/Bioconductor package for functional exploration of aligned next-generation sequencing reads. Bioinformatics 26:2902–2903CrossRefPubMedPubMedCentralGoogle Scholar
Morgan M, Anders S, Lawrence M, Aboyoun P, Pagès H, Gentleman R (2009) ShortRead: a Bioconductor package for input, quality assessment and exploration of high-throughput sequence data. Bioinformatics 25:2607–2608CrossRefPubMedPubMedCentralGoogle Scholar
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map (SAM) format and SAMtools. Bioinformatics 25:2078–2079CrossRefPubMedPubMedCentralGoogle Scholar
Song L, Axtell MJ, Fedoroff NV (2010) RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr Biol 20:37–41CrossRefPubMedGoogle Scholar
Iki T, Yoshikawa M, Nishikiori M, Jaudal MC, Matsumoto-Yokoyama E, Mitsuhara I, Meshi T, Ishikawa M (2010) In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Mol Cell 39:282–291CrossRefPubMedGoogle Scholar
Tsuzuki et al. (2016) The result of analysis of Marchantia miRNA appeared as a paper, Plant Cell Physiol 57:359–372Google Scholar