Tandem affinity purification of miRNA target mRNAs (TAP-Tar).

Nucleic acids research (2009-12-04)
Nora Nonne, Maya Ameyar-Zazoua, Mouloud Souidi, Annick Harel-Bellan
RESUMEN

MicroRNAs (miRNAs) bind to Argonaute proteins, and together they form the RISC complex and regulate target mRNA translation and/or stability. Identification of mRNA targets is key to deciphering the physiological functions and mode of action of miRNAs. In mammals, miRNAs are generally poorly homologous to their target sequence, and target identification cannot be based solely on bioinformatics. Here, we describe a biochemical approach, based on tandem affinity purification, in which mRNA/miRNA complexes are sequentially pulled down, first via the Argonaute moiety and then via the miRNA. Our 'TAP-Tar' procedure allows the specific pull down of mRNA targets of miRNA. It is useful for validation of targets predicted in silico, and, potentially, for discovery of previously uncharacterized targets.

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Millipore
ANTI-FLAG® M2 Affinity Gel, purified immunoglobulin, buffered aqueous glycerol solution
Sigma-Aldrich
Anti-Mouse IgG (Fab specific)–Peroxidase antibody produced in goat, affinity isolated antibody, buffered aqueous solution