MEGAshortscript™ T7 High Yield Transcription Kit from Applied Biosystems

in vitro transcription is a commonly used method for producing vast copy numbers of RNA. However, using conventional methods, transcription of small templates often results in low mass yields of RNA. Short transcripts require many more initiation events to synthesize a given mass of product. Therefore, since the initiation step is the rate-limiting step in transcription reactions, the yield of product from short templates is often low.

The MEGAshortscript™ T7 Kit from Applied Biosystems is designed for high yields of in vitro transcribed RNA products, in the 20-500 nucleotide range. It contains reagents for 25 in vitro transcription reactions (each 20 ul). Included are the T7 enzyme mix, the 10X reaction buffer, a set of nucleotides, TURBO DNase, and for stopping the reaction, either ammonium acetate stop solution or gel loading buffer II, dependent on the method used to purify the RNA. If the ammonium ions will interfere with downstream applications, the stop solution can be replaced with 3 M sodium acetate. Materials which are not provided by the manufacturer are a DNA template containing a T7 promoter, reagents for purification of the RNA, like phenol/chloroform-ethanol or a spin column based technique like the MEGAclear kit from Applied Biosystems.

I used this kit frequently for the synthesis of shorter RNAs (up to 500 nucleotides, using PCR templates) for naked RNA transfection experiments and activity assays where an RNA template is necessary, like in vitro splicing assays or reverse transcriptase activity assays.

There are several things I liked about this kit. First, different template types can be used without any changes in your protocol. However, the best transcription efficiency is usually obtained with linearized plasmid, followed by PCR templates. Synthetic oligonucleotide templates can also be used but are usually the least efficiently transcribed templates. I also enjoyed the fact that the RNA in the reaction is always protected from ribonucleases by a broad spectrum RNase inhibitor present in the T7 enzyme mix. Lastly, the protocol is very straightforward, and contains the necessary troubleshooting paragraph as well as additional protocols for designing templates with a T7 promoter.

The assembly of the reaction is easy and fast, however, one important detail is that the reaction mix needs to be prepared at room temperature and the reagents need to be added in the order as listed in the protocol. The reaction mix gets incubated for 2-4 hr at 37ºC using a PCR machine. After the incubation, the sample can be optionally treated with DNase to remove the DNA template. The reaction gets terminated by performing any of the possible purification procedures recommended by the manufacturer.

Overall, I think this kit is working great for in vitro transcribing short templates. It is easy to set up and to use and met my expectations.