Identification and Quantification of Small RNAs

Methods Mol Biol. 2021:2200:225-254. doi: 10.1007/978-1-0716-0880-7_11.

Abstract

RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing is guided by small non-coding RNAs (sRNAs) with the length of 21-24 nucleotides (nt) that are loaded into Argonaute (AGO) to repress expression of target loci and transcripts through transcriptional or posttranscriptional gene silencing mechanisms. Identification and quantitative characterization of sRNAs are crucial steps toward appreciation of their functions in biology. Here, we developed a step-by-step protocol to precisely illustrate the process of cloning of sRNA libraries and correspondingly computational analysis of the recovered sRNAs. This protocol can be used in all kinds of organisms, including Arabidopsis, and is compatible with various high-throughput sequence technologies such as Illumina Hiseq. Thus, we wish that this protocol represents an accurate way to identify and quantify sRNAs in vivo.

Keywords: Computational analysis; High-throughput sequencing; Library construction; RNA silencing; Small RNA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis Proteins / biosynthesis
  • Arabidopsis Proteins / genetics
  • Arabidopsis* / genetics
  • Arabidopsis* / metabolism
  • Argonaute Proteins / biosynthesis
  • Argonaute Proteins / genetics
  • Gene Expression Regulation, Plant*
  • High-Throughput Nucleotide Sequencing*
  • RNA, Plant* / biosynthesis
  • RNA, Plant* / genetics
  • RNA, Small Untranslated* / biosynthesis
  • RNA, Small Untranslated* / genetics

Substances

  • Arabidopsis Proteins
  • Argonaute Proteins
  • RNA, Plant
  • RNA, Small Untranslated