Deep sequencing analysis of nucleolar small RNAs: RNA isolation and library preparation

Baoyan Bai, Marikki Laiho

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The nucleolus is a subcellular compartment with a key essential function in ribosome biogenesis. The nucleolus is rich in noncoding RNAs, mostly the ribosomal RNAs and small nucleolar RNAs. Surprisingly, also several miRNAs have been detected in the nucleolus, raising the question as to whether other small RNA species are present and functional in the nucleolus. We have developed a strategy for stepwise enrichment of nucleolar small RNAs from the total nucleolar RNA extracts and subsequent construction of nucleolar small RNA libraries which are suitable for deep sequencing. Our method successfully isolates the small RNA population from total RNAs and monitors the RNA quality in each step to ensure that small RNAs recovered represent the actual small RNA population in the nucleolus and not degradation products from larger RNAs. We have further applied this approach to characterize the distribution of small RNAs in different cellular compartments.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages231-241
Number of pages11
DOIs
StatePublished - 2016

Publication series

NameMethods in Molecular Biology
Volume1455
ISSN (Print)1064-3745

Keywords

  • Deep sequencing
  • Library preparation
  • Nucleolus
  • Small RNA purification

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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