title: Deep Sequencing Analysis of Nucleolar Small RNAs: RNA Isolation and Library Preparation.
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.
authors: Department of Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, and Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway.
authors: Baoyan
authors: B
authors: Bai
authors: Department of Radiation Oncology and Molecular Radiation Sciences, Johns Hopkins University School of Medicine, 1550 Orleans Street, CRB2, Room 444, Baltimore, MD, 21287, USA. mlaiho1@jhmi.edu.|Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA. mlaiho1@jhmi.edu.
authors: Marikki
authors: M
authors: Laiho
mesh_terms: D002466:Cell Nucleolus / Q000235:genetics; D018076:DNA, Complementary; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D020537:RNA, Small Nucleolar / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D018076:DNA, Complementary; D020537:RNA, Small Nucleolar
keywords: Deep sequencing; Library preparation; Nucleolus; Small RNA purification