Advancing Epigenetics Towards Systems Biology

Native Purification and Analysis of Long RNAs

Isabel Chillón1, Marco Marcia1,4, Michal Legiewicz1, Fei Liu1, Srinivas Somarowthu1, Anna Marie Pyle1,2,3

Introduction

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.

PDF version

Isabel Chillón1, Marco Marcia1,4, Michal Legiewicz1, Fei Liu1, Srinivas Somarowthu1, Anna Marie Pyle1,2,3

1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA
2 Howard Hughes Medical Institute, Chevy Chase, Maryland, USA
3 Department of Chemistry, Yale University, New Haven, Connecticut, USA
4 European Molecular Biology Laboratory, Grenoble Outstation, France

Corresponding author: Marco Marcia
Email feedback to: This email address is being protected from spambots. You need JavaScript enabled to view it.

Isabel Chillón, Marco Marcia<sup>1,4</sup>, Michal Legiewicz, Fei Liu, Srinivas Somarowthu, Anna Marie Pyle<sup>1,2,3</sup>
Mon, May 29th 2017- Wed, May 31st 2017

After last year's successful kick-off meeting, the EpiGeneSwiss meeting will take place again in Weggis, Switzerland. This year we have added one day in addition to accommodate more topics and more t...

Mon, Jun 19th 2017- Wed, Jun 21st 2017

The focus of the 2017 IMB Conference is on “Gene Regulation by the Numbers: Quantitative Approaches to Study Transcription”. The conference will explore the latest findings and technological developme...

Tue, Jul 11th 2017- Thu, Jul 13th 2017

The MRC Integrative Epidemiology Unit at the University of Bristol invites all professionals interested in aetiological epidemiology and Mendelian randomization to attend our conference on the subject...

Wed, Aug 30th 2017- Fri, Sep 1st 2017

The nucleosome is the fundamental building block of chromatin organisation and genome function. The 20th anniversary of the high resolution nucleosome structure marks a milestone for bringing together...

LAST EVENTS

EpiGeneSys Final
Meeting in Paris

Thur. 11 February 2016 - Sat. 13 February 2016

More than 280 scientists attended the fifth Annual Meeting of EpiGeneSys. The conference kicked off with a talk by coordinator Geneviève Almouzni, Director of the Research Center at the Institut Curie, highlighting the achievements of the network over more than five years...

Maison des océans - Paris Read more

PAST EVENTS

The Non-Coding Genome ...

December 3-4 th, 2015

The last training workshop of the EpiGeneSys network

Hotel Mediterraneo - Rome, Italy Read more

Paris / TriRhena Chromatin Club

July 9th, 2015

...exciting talks and network with members of the Chromatin community!

... An EpiGeneSys TAB workshop

June 11st-12nd , 2015

... learn about current approaches to single cell epigenetics and to meet up and network with...

Montpellier, FranceRead more

Latest publications

2017-04-30

The impact of rare and low-frequency genetic variants in common disease.

Read more
2017-04-25

Stable Polycomb-dependent transgenerational inheritance of chromatin states in Drosophila.

Read more
2017-04-25

Stable Polycomb-dependent transgenerational inheritance of chromatin states in Drosophila.

Read more