Top Ten Tips for Successful Transcription

The most common uses of in vitro transcribed RNA are for probe synthesis, in vivo expression studies, and RNA transcript structural analysis. The application will usually determine whether transcription should be performed in large or small scale reactions. Here are some tips for successful transcription experiments.

1. Preparing the DNA template for transcription
While PCR products and oligonucleotides can typically be used directly in transcription reactions without problems, plasmid preparations may need to be cleaned up. This is due to the common use of RNase to remove bacterial RNA during plasmid preparation. The RNase can still be present in the purified plasmid sample and should be destroyed by proteinase K digestion, followed by phenol/chloroform extraction.

2. Choosing an RNA polymerase
There are 3 RNA polymerases that catalyze RNA synthesis: T7, T3, and SP6 RNA Polymerases. Each has a unique associated promoter sequence and distinct optimal buffering conditions under which it functions maximally—for example, optimal salt concentrations vary among the three polymerases. Thus, it is important to use the correct polymerase-associated buffer. Note that some manufacturers alter the salt concentration within the enzyme storage buffer so that 3 polymerases can be used with one universal buffer. While all 3 polymerases are similarly effective at transcribing RNA, differences in stability as well as pH and salt tolerance have led to T7 RNA polymerase being considered the strongest/best polymerase of the three.

3. Using PCR products as templates
While plasmids engineered to contain RNA polymerase promoters have been the traditional templates for transcription, PCR products can function as templates for transcription as well. A promoter can be added to the PCR product by including the promoter sequence at the 5’ end of either the forward or reverse PCR primer. These bases become double-stranded promoter sequences during the PCR reaction.

4. Synthesizing very short (<30) transcripts
Two oligonucleotides can also be used to create short transcription templates. Two complementary oligonucleotides containing a phage promoter sequence are simply annealed to make a double-stranded DNA template. Only part of the DNA template—the –17 to +1 bases of the RNA polymerase promoter—needs to be double-stranded. It may be more economical, therefore, to synthesize one short and one long oligonucleotide, generating an asymmetric hybrid.

5. Determining whether you will get sense or antisense transcript
If the RNA is to be used as a probe for hybridization to messenger RNA (e.g., Northern blots, in situ hybridizations, and ribonuclease protection assays), complementary antisense transcripts are required. In contrast, sense strand transcripts are used when performing expression, structural, or functional studies or when constructing a standard curve for RNA quantitation using an artificial sense strand RNA.

The +1 G of the RNA polymerase promoter sequence in the DNA template is the first base incorporated into the transcription product. To make sense RNA, the 5’ end of the coding strand must be adjacent to or just downstream of, the +1 G of the promoter. For antisense RNA to be transcribed the 5' end of the noncoding strand must be adjacent to the +1 G. If the insert is in a vector, the vector should be linearized downstream from the promoter and the inserted sequence to be transcribed.

6. Radiolabeling RNA transcripts
Use small scale transcription reactions with relatively low nucleotide concentrations (0.5 mM each) when you need to radiolabel RNA to high specific activity. Higher nucleotide concentrations are not necessary in these reactions, since the low concentration of radiolabeled or modified nucleotide present effectively limits the total yield of the reaction. The total concentration of the limiting nucleotide (labeled/modified and unlabeled) should be at least 3 µM for efficient synthesis of full length RNA transcripts of <400 nt (more will be needed to synthesize longer transcripts). Ambion MAXIscript® Kits are ideal for synthesizing RNA labeled to high specific activity.

7. Transcribing large mass amounts of RNA
Large scale in vitro transcription reactions can produce up to 120−180 µg RNA/µg template in a 20 µL reaction. The Ambion MEGAscript™ Kit uses patented technology that allows the phage RNA polymerases to remain active at high nucleotide concentrations that would ordinarily inhibit the enzyme. Yields from these large scale reactions are typically 10 to 50 times higher than those possible with conventional transcription reactions (without any limiting nucleotide). Reaction conditions (e.g., the type of nucleotide salt, type and concentration of salt in the transcription buffer, enzyme concentration, and pH) are all optimized not only for each polymerase but also for the entire set of components. Optimal yields can be achieved only under these conditions.

8. Amplifying RNA >3000X
Applications such as array analysis require large amounts of RNA sample. This challenge is compounded by the frequent need to profile the RNA population from very small samples. It is therefore sometimes necessary to amplify RNA by transcription from cDNA templates synthesized from the original RNA sample. Large scale transcription reactions can linearly amplify RNA >3000 fold. Ambion MessageAmp™ aRNA Kits are complete kits for aRNA amplification based on the patented Eberwine method. Incorporating MEGAscript™ high yield transcription technology, these kits include all necessary reagents for first-strand cDNA synthesis, RNase H digestion, second-strand synthesis, cDNA purification, in vitro transcription (amplification), and aRNA purification.

9. Dealing with troublesome templates
The most common ways to alleviate premature transcription termination or lack of transcription are:
• Clean up the DNA template
• Increase the concentration of the limiting nucleotide
• Lower the incubation temperature of the reaction (and increase the incubation time)
• Use a different polymerase

10. Keeping RNA intact after transcription
RNA should be stored frozen, at −20ºC or colder, ideally in an RNase-free, low pH buffer containing divalent cation chelating agents. There are several Ambion RNA storage solutions available that meet these criteria. If the RNA sample will be used frequently, the stock tube should be aliquoted to prevent shearing due to frequent freeze-thawing.

There are a family of Ambion transcription kits specialized for different applications. Kits for RNA amplification and RNA storage solutions are also available.