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Practical Tips for In Vitro
Transcription
by Lorianne Martin, M.S.
Generating Full-length Transcripts
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 - the True Story
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. Detailed instructions for calculating specific
activity are included in the MAXIscriptà manual and in Ambion's Technical
Bulletin #174: Determining RNA Probe Specific
Activity.
pGEM® and pBluescript® are registered
trademarks of Promega and Stratagene, respectively.
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