Related Experiment Video
Updated: Nov 19, 2025
Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
Published on: May 5, 2018
A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol
Dimitrios G Anastasakis1, Alexis Jacob1, Parthena Konstantinidou2
1Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, 20892 MD, USA.
This study introduces fluorescence-based photoactivatable ribonucleoside enhanced CLIP (fPAR-CLIP), a faster and more sensitive method for mapping protein-RNA interactions. This technique avoids radioactivity and streamlines the process for analyzing gene regulation.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Crosslinking and immunoprecipitation (CLIP) methods are crucial for studying protein-RNA interactions.
- Photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) uses photoreactive nucleosides for crosslinking, enabling nucleotide-resolution mapping of RNA binding sites.
- Existing PAR-CLIP protocols can be time-consuming and involve radioactivity.
Purpose of the Study:
- To develop a streamlined, non-radioactive protocol for PAR-CLIP.
- To enhance the sensitivity and efficiency of mapping protein-RNA interactions.
- To facilitate the analysis of posttranscriptional gene regulatory networks.
Main Methods:
- Developed fluorescence-based PAR-CLIP (fPAR-CLIP).
- Utilized in vivo labeling with photoreactive nucleosides (4SU or 6SG).
- Employed direct adapter ligation to the 3' end of crosslinked RNA on immobilized ribonucleoproteins, followed by cDNA conversion without size fractionation.
Main Results:
- The fPAR-CLIP protocol eliminates the need for radioactivity.
- The method significantly reduces experimental time by half (to 2 days).
- Achieved a 10-100 fold increase in sensitivity compared to conventional methods.
Conclusions:
- fPAR-CLIP offers a more efficient and sensitive approach to study protein-RNA interactions.
- This streamlined protocol facilitates the investigation of gene regulatory networks.
- The method provides nucleotide-resolution mapping of RNA binding protein sites.
