Files and links (1)
Accepted (AM)Open Access (License Unspecified), Open
Book chapter
Native Purification and Analysis of Long RNAs
Methods in enzymology
01 Jan 2015
PMID: 26068736
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation-renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2'-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing.
Metrics
Details
- Title
- Native Purification and Analysis of Long RNAs
- Creators
- Isabel Chillon - Yale UniversityMarco Marcia - Yale UniversityMichal Legiewicz - Yale UniversityFei Liu - Yale UniversitySrinivas Somarowthu - Yale UniversityAnna Marie Pyle - Yale University
- Contributors
- S A Woodson (Editor)FHT Allain (Editor)
- Publication Details
- Methods in enzymology
- Series
- Methods in Enzymology
- Publisher
- Elsevier; SAN DIEGO
- Number of pages
- 35
- Grant note
- Howard Hughes Medical Institute R01GM050313 / NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS) R01GM50313; R01 GM050313 / NIGMS NIH HHS; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS)
- Resource Type
- Book chapter
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology
- Web of Science ID
- WOS:000405656400002
- Scopus ID
- 2-s2.0-84935004525
- Other Identifier
- 991020837832804721
UN Sustainable Development Goals (SDGs)
This publication has contributed to the advancement of the following goals:
InCites Highlights
Data related to this publication, from InCites Benchmarking & Analytics tool:
- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemical Research Methods
- Biochemistry & Molecular Biology