Poly(A) Tailing Kit From Applied Biosystems

Polyadenylation plays an important role in the stabilization of RNA in eukaryotes and enhances the efficiency of translation initiation. In vitro transcribed RNA is generated without a poly(A) tail. Using a poly(A) tailing kit to generate tailed RNA might be an advantage over using untailed RNA in transfection or micro-injection experiments where enhanced translation may be seen due to increased stability and therefore, increased translation efficiency.

The Poly(A) Tailing Kit from Applied Biosystems contains the reagents for 25 reactions, including the poly(A) polymerase, buffer, ATP solution, and all the other necessary reagents to perform this simple reaction. The kit is available in two versions, with or without a user manual, which can be downloaded from an online source. The protocol is easy to follow and detailed. The manual includes a detailed troubleshooting paragraph and also includes a paragraph on using denaturing agarose gel electrophoresis for quality control purposes after the reaction.

In my hands, the kit was used for performing poly(A) tailing reactions on in vitro transcribed mRNAs or RNA 3¡¯ end regions which needed to be poly(A) tailed for downstream applications such as transfection or enzyme activity tests.

The kit's reagents are designed to add a ¡Ã 150 base poly(A) tail to RNA transcripts. It is optimized for use with Ambion¡¯s mMessage mMachineTM High Yield Capped RNA Transcription Kit; however, I used different in vitro transcription kits (MEGAscript¢â and MEGAshortscript¢â from Ambion/Applied Biosystems) and the poly(A) tailing reaction worked very well for each of the products generated. One nice feature of the kit is the flexibility regarding the length of the poly(A) tail synthesized. To generate tails shorter than ~ 150 bases, the polymerase gets diluted in buffer, and therefore, less enzyme gets used in the reaction. For some transcripts, I had a hard time getting a good tail, however, by increasing the amount of enzyme added (up to 16 units, standard is 8 units) to the reaction I got great poly(A) tailed products. The setup of the reaction is very simple. All the reagents, nuclease-free water, reaction buffer, magnesium-chloride and ATP are added to the template of choice. Since at this point the enzyme is not added yet, a negative control can be taken out of the reaction mix, a step I always performed. By adding the enzyme, the reaction is started. As reaction enzyme E. coli Poly(A) Polymerase I (E-PAP), a terminal adenyltransferase with high specificity for ATP and RNA substrates, is used. After an incubation period of 1 hr at 37¨¬C the reaction is terminated by placing the reaction on ice, or if preferred, and for a lot of downstream applications necessary, a clean-up step using either a precipitation approach or a clean-up kit with spin columns is performed. Most of the time, I used the MEGAclear¢â kit from Applied Biosystems with great success.

Everything taken together, this is a great kit for fast, simple and efficient tailing reactions.