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Epicentre Forum 1 (4)
AmpliScribe T7, SP6, and T3 Transcription Kits are
designed for high yield in vitro synthesis of RNA ranging in size
from as small as 25 nucleotides to multi-kilobases. The unique enzyme
formulations in the AmpliScribe Kits enable researchers to obtain
RNA yields 10-30 times higher than those possible with conventional transcription
kits. In addition, AmpliScribe kits can be used to generate capped transcripts.
Answers to some of the questions that Epicentre's Technical Services
Department has received concerning AmpliScribe Transcription Kits
are given below. If you have any other questions related to AmpliScribe Kits
or other products, please contact Epicentre's Technical Services.
I want to synthesize 5 different RNAs, ranging
in size from 150 bases to 8 kilobases. What yields can I expect?
Transcripts throughout this size range can be produced
using an AmpliScribe Transcription Kit. As a benchmark, the AmpliScribe T7
Control Template yields about 150 µg of a 1.4 kb RNA transcript
in a 20 µl AmpliScribe reaction in 2 hr, or about 900 molecules
of RNA per molecule of DNA template. The yield from other templates will
vary somewhat, depending on the DNA sequence and the size of the RNA
it encodes. Because initiation is the rate-limiting step in most transcription
reactions, highest mass (µg) yields per reaction are obtained using
templates encoding larger transcripts. Smaller transcripts will result
in lower mass, but higher molar yields. For example, yields of 45-60 µg
of a 63-base active ribozyme were obtained per 20 µl reaction in
2 hr using an AmpliScribe T7 Kit;1 thus,
greater than 18,500 RNA molecules were obtained per template molecule
in these transcription reactions.
What yields can I expect for capped transcripts?
For synthesis of capped RNA using a cap analog, the concentration
of GTP is reduced compared to standard AmpliScribe reactions in
order to favor incorporation of the cap analog. Thus, the yield of RNA
from these reactions will be lower than that from standard reactions.
Still, higher yields of capped transcripts will be obtained using AmpliScribe Kits
than using conventional kits. For example, we have observed yields of
30 µg to 60 µg per 20 µl reaction, with approximately
50% capping, when transcribing the AmpliScribe T7 Control Template.
Of course, yields will vary depending on the template.
What can I do to assure that a single full-length
transcript is obtained?
Prior to the transcription reaction, the template should
be linearized using a restriction enzyme that generates a 5´-overhang
or a blunt end. 3´-overhangs generated by enzymes like Pst I can
serve as false transcription start sites, resulting in spurious products.2 Also,
carefully check that the restriction enzyme digestion of the template
has gone to completion. Incomplete digestion may result in "run-on" transcription
because the RNA polymerase will transcribe continuously around a circular
plasmid template.
I would like to generate transcripts from a small
oligonucleotide carrying the T7 promoter rather than from a plasmid.
Is this possible?
Yes, transcription from oligonucleotides carrying a phage
promoter is possible, but yields may be reduced relative to yields from
plasmid templates. Ideally, such templates should be completely double-stranded
molecules. Although transcription is possible from oligonucleotide templates
in which only the promoter portion is double-stranded,3 the
single-stranded regions of the template, as well as other single-stranded
DNA in a reaction mixture may be transcribed in a promoter-independent
fashion, resulting in non-specific transcripts. Therefore, we recommend
using only completely double-stranded templates for most applications.
I am synthesizing a very small RNA, and obtaining
products that differ in size by a few bases from my expected product.
What could be happening?
There is often heterogeneity in both the initiation, and
termination of transcription when using phage polymerases. For example,
researchers working with SP6 RNA polymerase have observed termination
of transcription at both the last and penultimate nucleotides of the
transcription template, as well as the addition of a 3´-terminal
non-encoded nucleotide.4 Others
have observed heterogeneity in the location of the start of transcription
with T7 RNA polymerase, with "start sites" in each of the final
three bases of the promoter.5
Although variability in transcript size due to imprecise
initiation and termination may occur for RNA of any size, size variability
is more obvious for small transcripts. Researchers synthesizing small
RNAs may notice a family of products differing in size by a few bases
after gel analysis of transcripts. However, even with this variability,
the majority of the transcripts will have the expected 5´- and 3´-
sequences, and the minority that have variable ends will likely be biologically
active.
Can reactions using AmpliScribe Kits be modified
to improve yields?
Epicentre's scientists have optimized variables for in
vitro transcription using AmpliScribe Kits with typical
templates to the point that it is difficult to improve yields. For
example, 75% of all of the NTPs in a standard 20 µl reaction
mixture are incorporated into RNA in 2 hr using the Control Template
provided with the AmpliScribe T7 Transcription Kit. Even higher
yields can be obtained by lengthening the reaction time to 3 hr. Reactions
longer than 3 hr are not recommended, as they could result in RNA degradation.
When synthesizing small (less than 300 bases), or difficult-to-transcribe
templates, yields may be improved by raising the temperature of the
reaction to 42°C.
What methods do you recommend for isolating my
RNA after transcription?
The AmpliScribe reaction contains large amounts
of NTPs, salts, and protein that may need to be removed before further
manipulation of the RNA. Although organic extraction and ethanol precipitation
may be used for RNA purification, we generally recommend an ammonium
acetate precipitation procedure6 for
transcripts longer than 100 bases. This procedure results in selective
precipitation of the RNA to remove DNA, NTPs, and protein. Virtually
100% of the RNA is recovered, and it is both pure and biologically active.
The ammonium acetate precipitation procedure is simple and eliminates
the need for both DNase I treatment and organic extraction. However,
since ammonium ions strongly inhibit T4 polynucleotide kinase, care should
be taken to remove all of the salt from the RNA prior to end-labeling
or similar reactions.
Can AmpliScribe Kits be used to synthesize high
specific activity radiolabeled RNA?
AmpliScribe Kits are optimized for the production
of large amounts of non-radioactive RNA, and generally are not recommended
for synthesis of radioactive RNA. Because of the high concentration of
NTPs used in our standard protocol, it is difficult to add enough of
a radioactive NTP to synthesize radiolabeled RNA of high specific activity.
Although, with a modified protocol, some of our customers have synthesized
extremely high specific activity RNA (in much lower yields than would
be obtained in a standard AmpliScribe reaction). Epicentre's T7,
SP6, and T3 RiboScribe™ Kits are preferable for synthesis of radiolabeled
RNA probes.
Can I substitute a high concentration of phage RNA
polymerase for the AmpliScribe enzyme solution in an AmpliScribe
reaction?
No. The AmpliScribe enzyme mix is specially formulated
to allow yields that are not possible with even the highest concentration
of the corresponding phage RNA polymerase.
- Hoffman and Johnson (1994) BioTechniques 17, 372.
- Schenbom and Mierendorf, (1985) Nucleic Acids Res. 13, 6223.
- Milligan et al. (1987) Nucleic Acids Res. 15, 8783.
- Nielson and Shapiro (1986) Nucleic Acids Res. 14, 5936.
- Melton et al. (1984) Nucleic Acids Res. 12, 7035.
- Epicentre Forum (1994) 1(2), 8.
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