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MessageAmp™ II-Biotin Enhanced:
A Single
Round aRNA Amplification Kit
Synthesize High Yields
of Biotinylated
aRNA
Ambion now offers an improved MessageAmp™ II Kit with
biotin-11-UTP. MessageAmp II-Biotin Enhanced provides
a complete single round amplification kit for biotin array analysis.
The biotin modified UTP and ratio of labeled to unlabeled UTP
were selected based on superior performance and aRNA yield. The
biotinylated nucleotide is supplied as a pre-mixed NTP at an
optimized ratio, providing convenient,
consistent labeling.
• Highest amplification in a single round—up
to 10,000-fold for most samples
• Enough aRNA for Affymetrix® GeneChip® analysis
from just 50 ng total RNA
• Optimized NTP mix containing
biotinylated UTP reduces set-up time and maximizes labeling
• Reduce costs by 45%
T7 RNA Polymerase Linear Amplification
is the Gold
Standard
Microarrays measure the expression levels of
thousands of genes. One limitation of this technology, however,
is the need for large amounts of labeled target RNA for hybridization
to the microarray. This limitation is overcome by using a
T7 linear amplification protocol commonly referred to as the “Eberwine
Method.” This method most accurately maintains the initial mRNA expression
profile and has been the gold standard for microarray target preparation [1].
Biotin, incorporated as biotinylated nucleotide triphosphates during the in
vitro transcription (IVT) step, is the preferred label for most commercial
microarray platforms. The highly specific and efficient binding of streptavidin-dye
conjugates (phycoerythrin,
Cy™ dyes) to biotinylated RNA/oligonucleotide probe hybrids is the basis of
highly sensitive fluorescence detection. Ambion has incorporated biotin labeling
reagents into the MessageAmp II Kit to provide a complete microarray aRNA preparation
kit, the MessageAmp II-Biotin Enhanced Kit.
Higher Yields of Labeled aRNA;
Only 1 Round of Amplification
Both biotin incorporation and further
optimization of the IVT reaction has resulted in increased
sensitivity of microarray transcript detection. The increased
sensitivity allows the use of less input RNA-50 ng of input
RNA in a single round of amplification provides enough aRNA
for array analysis. Amplified RNA yields derived from nine
different tissue and cell sources and four different input
amounts are shown in Figure 1. While length of the aRNA synthesized
will largely depend on RNA sample quality, the cDNA and IVT
reaction conditions in this kit produce longer biotinylated
aRNA compared to the market leader. Figure 2 shows comparable
Agilent® bioanalyzer scan results from labeled
aRNA produced with MessageAmp II-Biotin Enhanced Kit
and unlabeled aRNA produced with MessageAmp II Kit. The resulting
improvements also give the MessageAmp II-Biotin Enhanced Kit
a higher number of Present calls than the market leader (Figure
3).
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Figure 1. aRNA Yield from Nine Different Sources. Four different amounts of total RNA from nine different sample types were amplified using the MessageAmp™ II-Biotin Enhanced Kit. Average aRNA yields from triplicate reactions are shown for 4 or 14 hr in vitro transcription (IVT) reactions. These data are useful for determining both the amount of total RNA needed to obtain enough labeled aRNA for array hybridization (typically ~10 µg) and the optimal length of IVT incubation. Note that there is a ~3-fold difference in aRNA yield between some samples. With most RNA sources, 50–100 ng of input total RNA amplified with the MessageAmp II-Biotin Enhanced Kit using a 14 hr IVT incubation will yield enough labeled aRNA for microarray hybridization.
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Figure
2. Electropherogram of Biotin-labeled
vs. Unlabeled aRNA. Identical HeLa cell total
RNA samples (1000 ng) were amplified with either the
MessageAmp™ II-Biotin Enhanced Kit
to produce biotin-labeled aRNA or the MessageAmp II Kit
to produce unlabeled aRNA. The IVT reactions were carried
out for 4 hr. Equivalent mass amounts from each reaction
were analyzed on an Agilent® bioanalyzer. The shift
in aRNA size from the MessageAmp II-Biotin Enhanced sample
results from the incorporation of biotin into the aRNA.
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Figure
3. Number of Present Calls Detected
on Affymetrix® Human Genome Focus Arrays. The
indicated amounts of total RNA from HeLa cells was amplified
in triplicate using either the MessageAmp™ II-Biotin Enhanced Kit
(MA II-Biotin) or another manufacturer’s aRNA labeling
kit (Kit A). Two IVT incubation times (4 and 14 hrs)
were tested with 1000 ng of total RNA input. Amplification
of as little as 50 ng total RNA with the MessageAmp II-Biotin Enhanced Kit
produced a comparable number of Present calls as 1000
ng input RNA that was amplified with Kit A. Furthermore,
the amplification reaction can be completed in one day.
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Hybridization of the biotinylated aRNA produced from the MessageAmp
II-Biotin Enhanced Kit to Affymetrix GeneChip Arrays
was used to assess the sensitivity and reproducibility of the
method and reagents. Increased sensitivity can be measured several
ways. One indication of sensitivity is the number of Present
calls calculated by the GeneChip Operating Software (Figure 3).
Additional analysis of the distribution of signal intensities
indicates that the new MessageAmp II-Biotin Enhanced Kit
produces a higher mean value. This means that a there was a higher
signal intensity across the array (Figure 4). This results in
more reproducible array results and increased sensitivity of
lower expressed transcripts.
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Figure
4. Higher Signal Intensity Distribution
on Affymetrix® Human Genome Focus Arrays. Distributions
of log2 signal intensity values were plotted for the
MessageAmp™ II-Biotin Enhanced Kit and
another manufacturer’s amplification kit (Kit A).
The median values for each of the MessageAmp II-Biotin Enhanced plots
were similar for total RNA inputs ranging from 50 to
1000 ng. In contrast, signal intensities produced by
Kit A at the 1000 ng total RNA input level was markedly
reduced. The box and whiskers plot above each histogram
indicate the median value (vertical center line), interquartile
range (left and right sides of box), and the 10% and
90% quantiles (ends of line). Each plotted distribution
represents the average log2 signal intensity of triplicate
target preparations and hybridizations. IVT reactions
were carried out for 4 or 14 hrs as indicated. The data
were normalized within each group using Robust Multichip
Average (RMA) (See sidebar, Data Analysis: Robust Multichip
Average (RMA) and Affymetrix Algorithms).
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Biotin Incorporation
Biotin-11-UTP serves as the label in the MessageAmp II-Biotin Enhanced Kit.
Only a single biotinylated NTP is used because studies at Ambion
and elsewhere [2] showed that in previous dual labeling experiments
using biotin-UTP and biotin-CTP, the signal contributed by biotin-CTP
was minimal, and relative signal intensity between genes was
not altered with the use of a single biotin UTP label.
While various linker arm sizes (e.g., Biotin-11-UTP, Biotin-16-UTP)
did not affect modified UTP incorporation into aRNA during IVT,
the longer linker arms slightly impeded subsequent purification
of the aRNA. Biotin-11-UTP provided the highest yields of aRNA.
The ratio of biotin UTP to unlabeled UTP was optimized to provide
the highest yield of aRNA and the highest signal:noise on Affymetrix
GeneChip Arrays. A fairly wide range of concentrations of Biotin-UTP
(25–60%) can be used for acceptable detection, whereas at lower
concentrations the signal drops dramatically, and at higher concentrations
both the noise increases and the aRNA yield decreases.
Ambion realizes that microarray users may prefer to continue
using their own biotin-labeling method, or to experiment with
different labeling conditions. To accommodate this we offer two
Biotin-UTP nucleotides (Biotin-11-UTP and Biotin-16-UTP) separately,
which can be used with the standard MessageAmp II Kit.
MessageAmp II-Biotin Enhanced Kit Format
The MessageAmp II-Biotin Enhanced Kit provides enough
reagents for 20 single round amplification reactions. It can
be used in combination with the MessageAmp II Kit for small samples
that require 2 rounds of amplification (MessageAmp II for the
first round and MessageAmp II-Biotin Enhanced in the
second round for labeling). Biotin-UTP is provided premixed with
the other rNTPS for convenience and consistent, optimized labeling.
Scientific Contributors
Robert Setterquist, Mike Wilson, Charles Johnson, Shika Agarwal,
Sharmili Moturi • Ambion, Inc.
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