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Rapidly Synthesize
Large Amounts of Capped RNA
•Convenient format
- premixed cap analog and nucleotides provided
•Optimized for highest
yield of capped RNA
•80% capping efficiency
•Complete with SUPERase•In™ RNase
inhibitor, DNase I, and positive control
• Ideal for microinjection,
in vitro or in vivo translation
The mMESSAGE mMACHINE® High
Yield Capped RNA Transcription Kits use Ambion's patented high
yield transcription technology. Each reaction synthesizes 10-50
times the yield of capped RNA obtained with conventional in
vitro transcription reactions.
The reaction is simple. DNA template
is mixed with the reaction components and incubated at 37°C
for up to 2 hours. Figure 1 shows a time course experiment
that plots yield of a 3.7 kb capped transcript over time. With
this template, the mMESSAGE mMACHINE reaction is complete in
only 30 minutes. In addition to synthesizing high yields of
capped RNA transcripts (Figure 1), mMESSAGE mMACHINE reactions
also produce full-length transcripts. Figure 2 shows an Agilent
bioanalyzer 2100 electropherogram of the 3.7 kb transcript.
The presence of a single, strong peak indicates the predominance
of full length transcript. This is also evident in Figure 3,
in which mMESSAGE mMACHINE reactions producing 1.7 kb, 3.7
kb and 9 kb transcripts were analyzed on a conventional denaturing
agarose gel.
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| Figure 1. mMESSAGE
mMACHINE Reaction Time Course. A
3.7 kb template was transcribed in 3 replicate mMESSAGE
mMACHINE reactions as per protocol. After the indicated
incubation times, each reaction was DNase treated as per
protocol and a preliminary quantitatation was performed
with RiboGreen® (Molecular Probes). Each
reaction was then purified using the MEGAclear™ Kit (Ambion) and quantitated by UV spectrophotometry. |
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| Figure 2. Electropherogram
of a mMESSAGE mMACHINE Reaction. A
3.7 kb template was used in a 20 µl mMESSAGE mMACHINE
reaction as per protocol and incubated for 30 min followed
by analysis using the Agilent 2100 bioanalyzer. |
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| Figure 3. Gel
Analysis of mMESSAGE mMACHINE Reactions. Templates
generating transcripts of sizes 1.7 kb, 3.7 kb and 9 kb
were used in a mMESSAGE mMACHINE reaction as per protocol.
One µg of each reaction was denatured using NorthernMax®-Gly
Sample Loading Dye and analyzed on a 1% agarose gel with
EtBr. |
Cap analog is premixed with GTP at a
ratio optimized for the best compromise between yield and
proportion of capped transcripts (80%). The cap analog:GTP
ratio can be
adjusted with the GTP Solution provided in the kit to increase
yields of long transcripts (>4 kb); in fact, RNA transcripts
as long as 23 kb have been synthesized with mMESSAGE mMACHINE.
The kit includes SUPERase•In
RNase Inhibitor. SUPERase•In
inhibits RNases A, B, C, 1 and T1 and offers the greatest
available
protection against RNases (see
data).
DNase I and a positive control DNA template are also included.
The RNA synthesized from a mMESSAGE mMACHINE reaction is ideal for in vitro
or in vivo translation studies.
Ambion also offers the mMESSAGE mMACHINE
T7 Ultra Kit for the generation of RNA transcripts capped with
Anti Reverse Cap Analog (ARCA). A base modification in this
cap analog generates RNAs with ARCA incorporated only in the
functional, translatable orientation, resulting in higher protein
yields. The mMESSAGE mMACHINE T7 Ultra Kit includes reagents
for both transcription and the addition of a poly(A) tail to
capped transcripts.
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