Poly(A) Tail-Length Assay Kit (Invitrogen™)

Newly reconfigured kit for increased efficiency: 76455 1 KT Poly(A) Tail-Length Assay Kit is a direct replacement for 76450 1 KT

• 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 (e.g. northern blotting)
• One kit includes optimized reagents for 5 G/I Tailing, 20 RT and 80 PCR reactions

Description:
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 which 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.

Description:
The Poly(A) Tail-Length Assay Kit uses four key steps to enable poly(A) tail-length determination (Fig. 1).

In Step 1, poly(A) polymerase adds a limited number of guanosine and inosine residues to the 3’-ends of poly(A)-containing RNAs.

In Step 2, the tailed-RNAs are converted to DNA through reverse transcription using the newly added G/I tails as the priming sites.

In 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.

Finally, in Step 4, the PCR products are separated on an agarose or polyacrylamide gel (Fig. 2).

Kit components:
Kit contains all necessary reagents for 5 G/I Tailing, 20 reverse transcription, and 80 PCR reactions.

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