Invitrogen™ Poly(A) Tail-Length Assay Kit

Descrizione

Poly adenylation of eukaryotic mRNAs plays a critical role in RNA metabolism, including stability, enhanced translation, nuclear export, and miRNA mediated gene regulation. The Poly(A) Tail-Length Assay Kit allows you to quickly measure the poly(A) tails of multiple mRNAs in only three hours. This kit utilizes a novel G/I extension technique that preserves the 3' end of RNAs and ensures accurate measurement of poly(A) tail-length. And all reaction components have been optimized so you get quality data quickly.

Features

• Measure the poly(A) tail of multiple RNAs in under 3 hrs using common lab equipment
• Accurate measurement of poly(A) tail length compared to other assays
• One kit includes all necessary reagents for 5 G/I tailing, 20 RT, and 80 PCR reactions

Technology

The Poly(A) Tail-Length Assay Kit uses four key steps to enable poly(A) tail-length determination (see figure).

• Step 1—poly(A) polymerase adds a limited number of guanosine and inosine residues to the 3'-ends of poly(A)-containing RNAs.
• Step 2—the tailed-RNAs are converted to DNA through reverse transcription using the newly added G/I tails as the priming sites.
• Step 3—PCR amplification products are generated using two primer sets. A gene-specific forward and reverse primer set designed upstream of the polyadenylation site (e.g., the 3'-UTR) is produced as a control for the gene-of-interest. The second set of primers uses the gene-specific forward primer and the universal reverse primer provided with the kit to generate a product that includes the poly(A) tails of the gene-of-interest.
• Step 4—the PCR products are separated on an agarose or polyacrylamide gel (see figure).

Selected references

• Parker, R. and Song, H. (2004) Nat Struct Mol Biol. 11 121-127.
• Brodersen, D.E. et al. (2008) Biochim Biophys Acta. 1779, 532-537.
• Wu, L., Fan J. and Belasco, J.G. (2006) Proc Natl Acad Sci U S A. 103, 4034-4039.
• Izaurralde, E. et al. (2009) RNA 15, 21-32.
• Martin, G. and Keller, W. (1998) RNA 4, 226-230.
• Gauss-Müller, V. et al. (2001) Nucleic Acids Res. 29 E57-7