RIP-Assay Kit for microRNA is optimized to immunochemically isolate endogenous miRNAs, mRNAs and RBPs within mRNP complexes. The kit is designed to isolate cellular miRNAs that being incorporated into the RISC and/or to isolate unique group of miRNAs that bind to specific group of mRNAs encoding functionally related proteins.

Approach 1: RISC immunoprecipitation to isolate miRNAs and their target mRNAs in a global manner.

Mammalian miRNAs associate with members of the Argonaute (AGO) family proteins, the core of the RISC, and bind to partially complementary sequences in the 3’ UTR of specific target mRNAs. Therefore, using an antibody against one of the RISC components, such as RIP-Certified Anti-EIF2C2/AGO2 Antibody provided from MBL, researchers can isolate miRNAs that are incorporated into the RISC. This approach gives a global view of which miRNAs are fully processed and incorporated into the RISC for binding to their specific target mRNAs.

Approach 2: RBP immunoprecipitation to isolate a unique group of miRNAs that bind to a specific group of mRNAs encoding functionally related proteins.

mRNAs encoding functionally related proteins are coordinately regulated during cellular processes such as proliferation, differentiation or drug treatment. RBPs and miRNAs constitute the primary regulators of eukaryotic post-transcriptional mRNA expression. When using RIP-Certified RBP Antibodies provided from MBL, researchers can isolate mRNAs that encode functionally related RNAs and proteins. Using the MBL specific RBP Immunoprecipitation approach, researchers can isolate functionally related miRNAs as well as their target mRNAs that encode functionally related proteins.

In the RIP-Assay protocol, mRNP complexes are isolated from cell extracts by immunoprecipitation with RIP-Certified RBP Antibodies provided from MBL. mRNAs and miRNAs are isolated from mRNPs using the proprietary buffers provided in this RIP-Assay Kit for microRNA. MBL provides customers with three different protocols to isolate mRNAs and miRNAs depending on the customer’s needs. One protocol can provide the best recovery of both mRNAs and miRNAs, and the other protocol is simpler and quicker but provides a more moderate recovery of mRNAs and miRNAs.

Once purified, the mRNAs and miRNAs present in the complexes are analyzed to identify the target mRNAs of miRNAs using various molecular biology tools such as RT-PCR, gene expression analysis based on a suitable microarray platform (Chip analysis), or direct sequencing