Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Axtell MJ (2013) Classification and comparison of small RNAs from plants. Ann Rev Plant Biol 64:137–159
Willmann MR, Poethig RS (2007) Conservation and evolution of miRNA regulatory programs in plant development. Curr Opin Plant Biol 10:503–511
Schauer SE, Jacobsen SE, Meinke DW, Ray A (2002) DICER-LIKE1: blind men and elephants in Arabidopsis development. Trends Plant Sci 7:487–491
Peláez P, Sanchez F (2013) Small RNAs in plant defense responses during viral and bacterial interactions: similarities and differences. Front Plant Sci 4:343
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 123–138
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 26:2902–2903
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 25:2607–2608
Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
Axtell MJ (2013) ShortStack: comprehensive annotation and quantification of small RNA genes. RNA 19:740–751
An J, Lai J, Lehman ML, Nelson CC (2013) miRDeep*: an integrated application tool for miRNA identification from RNA sequencing data. Nucleic Acids Res 41:727–737
Kozomara A, Griffiths-Jones S (2014) miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42:D68–D73
Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26
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 25:2078–2079
Song L, Axtell MJ, Fedoroff NV (2010) RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr Biol 20:37–41
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 39:282–291
Tsuzuki et al. (2016) The result of analysis of Marchantia miRNA appeared as a paper, Plant Cell Physiol 57:359–372
Acknowledgments
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Tsuzuki, M., Watanabe, Y. (2017). Profiling New Small RNA Sequences. In: Kovalchuk, I. (eds) Plant Epigenetics. Methods in Molecular Biology, vol 1456. Humana Press, Boston, MA. https://doi.org/10.1007/978-1-4899-7708-3_14
Download citation
DOI: https://doi.org/10.1007/978-1-4899-7708-3_14
Published:
Publisher Name: Humana Press, Boston, MA
Print ISBN: 978-1-4899-7706-9
Online ISBN: 978-1-4899-7708-3
eBook Packages: Springer Protocols