6 reagents selected for iCLIP (including pre-adenylated linker L3)

miRNA - circulating

Individual-nucleotide resolution UV CrossLinking and ImmunoPrecipitation (iCLIP) enables the identification of protein-RNA interactions. The original CLIP protocol described by Jernej Ule et al. in 2003 (1) has evolved significantly (2, 3), and the current iteration of the iCLIP protocol requires a core set of oligonucleotides and enzymes.

iCLIP protocol

iCLIP protocol. Source: Bluewhale22 (http://commons.wikimedia.org/wiki/File%3AICLIP.JPG).

iCLIP is a powerful tool to perform transcriptome wide mapping of the binding sites of an RNA-binding proteins.

Nevertheless, this method requires high level of expertise and optimised reagents to obtain optimal results. When discussing this technique (which I have to admit was relatively unclear to me) with Mark,  my colleague and Molecular Biology Manager at tebu-bio, I have realized that an attractive “iCLIP bundle” based on this protocol was possible.

Here are 6 optimized reagents, including an iCLIP preadenylated linker, that previously had to be custom ordered.

Ideal “iCLIP” bundle:

Here are Mark’s suggestions:

  1. iCLIP Oligo (3 x 3 nmol; TriLink Biotechnologies, cat. nr 040O-30050-9)
  2. RNASE I (EpiCentre-Illumina) (cat. nr 035N6901K)
  3. T4 RNA Ligase 2 (EpiCentre Illumina, cat. nr 035LR2D1132K) – ligates the preadenylated 5´ ends of DNA or RNA are ligated to the 3´ ends of RNA
  4. Proteinase K (EpiCentre Illumina, cat. nr 035MPRK092)
  5. MonsterScript RT Kit (EpiCentre Illumina, cat. nr 035MS041050) –  EpiCentre’s answer to SuperScript III
  6. CircLigase II ssDNA Ligase (EpiCentre-Illumina, cat. nr 035CL9021K) –  circularizes ssDNA templates having a 5´-phosphate and a 3´-hydroxyl group in the absence of a complementary sequence

These 6 reagents can be used in combination with our antibodies optimized for IP.

 Want to know more about iCLIP?

You might like to access initial publications describing the initial iCLIP protocol:

  1. Ule J. et al. “CLIP Identifies Nova-Regulated RNA Networks in the Brain” (2003) Science, Vol. 302 no. 5648 pp. 1212-1215. DOI: 10.1126/science.1090095
  2. Ule J. et al. “CLIP: A method for identifying protein–RNA interaction sites in living cells” (2005) Methods, Vol. 37, Issue 4, pp. 376–386. DOI: 10.1016/j.ymeth.2005.07.018.
  3. Konig et al. “iCLIP – Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution” (2011) J Vis Exp., Vol. 50. pii: 2638. DOI: 10.3791/2638

Feel free to leave your comments, tips or recommendations you’d like to share with other researchers for iCLIP procedures!

Philippe Fixe, PhD
Written by Philippe Fixe, PhD
Philippe Fixe is a former Marketing Manager at tebu-bio, passionate about innovation and R&D in Life sciences, Biotechnology, Medical research, Drug discovery, and also a keen photographer.