Technical Bulletin: #182

Most DNA templates synthesized with Ambion's transcription kits generate full-length transcripts without any optimization. However, some templates may generate prematurely terminated products manifested as smaller discrete bands, or smears/degraded product. For blot hybridizations, full-length RNA probes are not generally required. For many other applications, however, it is crucial that transcription proceeds to the end of the template generating transcripts of all one size (e.g. NPAs, in vitro translation studies, and structural analyses). The two most common causes of failed or poor transcription reactions are inhibitors in the labeled nucleotide, and poor quality DNA template. A set of simple experiments should be done to determine the cause of failed transcription reactions; the accompanying flow chart outlines this process. Below are listed some additional strategies that can be used to increase the proportion of full-length products from problematic transcription reactions.

Increasing the concentration of the limiting nucleotide
Transcription reactions that are done with the minimum concentration of labeled nucleotide may produce prematurely terminated transcripts because of insufficient nucleotide concentration. Increasing the concentration of the limiting nucleotide will often improve the yield of full length transcripts.

Lowering the incubation temperature of the reaction
Typically, transcription reactions are performed at room temperature or at 37°C. Lowering the temperature to ~16°C or even 4°C can sometimes improve transcription. It is believed that lower reaction temperatures slow the polymerase's progression, thereby preventing it from being displaced by secondary structure or a string of one specific nucleotide. (Picture a toy train flying around a bend at full speed vs. slowly chugging around the same curve.)

Use a different polymerase
The three RNA polymerases commonly used for in vitro transcription may transcribe a given template somewhat differently from each other. This differential ability to transcribe a given template sequence can be exploited when transcription will not proceed to completion. Changing the polymerase promoter sequence in front of a transcription template is a somewhat labor intensive fix as it may require designing PCR primers or subcloning. Ambion has vectors and primers that simplify the process. The pTRIPLEscript family of vectors have SP6, T7, and T3 promoters arranged in tandem, and these vectors are particularly useful for subcloning transcription templates. Alternatively, Ambion's no-cloning promoter addition kit, Lig'nScribeÃ, can be used to change the promoter site of DNA fragments that can be PCR amplified.

Specific activity is measured in cpm/µg; it reflects the degree to which a molecule is labeled with radioactive nucleotides. The specific activity of transcription products is determined by the ratio of the labeled to unlabeled nucleotide present in the reaction. High specific activity probes are more sensitive than lower specific activity probes.

There is a trade-off between specific activity, and yield of full-length transcript.
When transcription reactions incorporate a labeled nucleotide, the concentration of that NTP is usually the limiting factor in the reaction. If the concentration of the "limiting nucleotide" is too low, the RNA polymerase may fail to produce full length transcripts. Supplementing the reaction with the unlabeled form of the limiting nucleotide will increase the yield of transcript and the proportion of full length probe, but it will reduce the specific activity of the transcripts. In other words, there will be fewer labeled nucleotides per molecule. Most transcription reactions will give a satisfactory yield of relatively high specific activity transcript when a final limiting nucleotide concentration of 3 µM is used (e.g. 5 µl of [- ³²P] UTP at 800 Ci/mmol and 10 mCi/ml; 12.5 µM in a 20 µl reaction).

Ambion's CU Minusà polymerase promoters work efficiently using as little as 1 µl of the highest specific activity labeled NTPs available.
If an application requires the maximum possible sensitivity, CU Minus promoters can reduce the premature termination of transcription often encountered in conditions of extremely low limiting nucleotide concentration - producing transcripts with 7.5X higher specific activity than those synthesized from traditional polymerase promoters. Transcription from CU Minus promoters can proceed to completion when the limiting nucleotide concentration is as low as 0.165 µM (This corresponds to 1 µl of [-32P] UTP at 3000 Ci/mmol and 10 mCi/ml). These promoters can be incorporated into pGEM´ or pBluescript´ constructs via PCR using Ambion's CU Minus primer pairs, or templates can be cloned into Ambion's exclusive CU Minus plasmid vectors, pDP18 and pDP19.

Calculating the specific activity of transcripts is usually not necessary.
It is always a good idea to check the percent incorporation of labeled nucleotide in transcription reactions that are radiolabeled; it is an easy way to get an idea of how well the reaction worked. Scintillation count aliquots of the reaction before and after removal of free nucleotides (by spin column or by TCA precipitation), and compare the cpm/µl. If 40% or more of the radiolabeled nucleotide was incorporated into RNA, it is probably not necessary to calculate either the mass amount of transcript produced, or the specific activity of the reaction products. If less than 40% incorporation is seen, the reaction or the radiolabeled nucleotide was not optimal and it may be prudent to calculate these values, and/or to look at the reaction products on a gel to be sure that enough full-length probe was made for your application.



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