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MessageAmp™ II-Biotin Enhanced Kit
Increase Signal and Detect More Genes on
Affymetrix® Arrays
• Complete kit for microarray sample
amplification and labeling
• Increase signal intensity resulting in increased Present calls
• Obtain enough amplified RNA for array analysis from a single round amplification
using 50 ng input RNA
• Reduce set up time and maximize labeling with optimized NTP mix that includes
biotinylated nucleotide
• Get optimal representation of gene expression profile with improved MEGAscript® IVT
technology
Performance Comparison: MessageAmp
II-Biotin Enhanced vs. Leading Competitor
Experimental Set-up. In
order to measure the difference between MessageAmp II-Biotin Enhanced and
a competitor’s Kit (Kit A), a series of experiments were
performed that demonstrated a significant improvement in performance
relative to the competing amplification process. The MessageAmp
II-Biotin Enhanced Kit yielded higher signal for 91.3%
of all genes on Affymetrix® Focus Arrays with equal or better
reproducibility as measured by the coefficient of variation (COV).
There was a resulting 8% increase in the number (4581 vs. 4230),
and percentage (52% vs. 48%) of Present calls. (The higher signal
was not due to overall increase in background, which was low
and the same for all arrays, which passed standard quality control
specifications.)
The two kits were compared in triplicate using
a common RNA source at 4 and 14 hr IVT reaction times, with 1000
ng of starting total RNA. There was an expectation that the overall
signal could be considerably different between kits, so the arrays
were not normalized or scaled, because the standard assumptions
for global normalization would be violated in this type of study.
To reduce technical variation, all arrays were run on the same
day by the same operator. In addition, to demonstrate an improvement
in performance on Affymetrix microarrays, a combination of ANOVA
analysis, hierarchical clustering, and graphics was used.
Analysis methods [1]. Because
the microarray is the end product of the amplification process,
the first step was microarray signal quantification. Background
subtraction, expression summary, and log transformation of gene
signals were carried out using the Robust Multichip Average (RMA;
developed by Irizarry et al. [2]) minus the quantile normalized
function for each Affymetrix Human Focus Array. Two-way ANOVA
analysis was carried out to resolve those genes that were differentially
expressed either between kit methods and/or IVT times.
Results. The
ANOVA analysis showed a large number of genes that differed significantly
between RNA amplification and labeling methods with less of an
impact when differences in IVT time were considered. A false
discovery rate (FDR) was calculated using a step-up approach
with a 0.05 significance value to compensate for multiple testing
errors. After the FDR procedure, 4766 out of 8732 genes differed
significantly between the MessageAmp II-Biotin Enhanced Kit
and Kit A, using a 14 hr IVT. Figure 1 shows the sources of variation
within the ANOVA model as measured by average mean square error,
but this gave no indication as to which kit is superior, only
that the choice of kit used had a major impact on the results.
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Figure 1. Source of Variance Shows Choice of Kit for RNA Amplification Matters. A two-way ANOVA analysis was carried out using Partek® Pro 6.1 (Partek Inc., St. Charles, MO) to determine whether genes were differentially expressed either between method and/or IVT times. The average mean square for each variable in the ANOVA model denotes the contribution of each variable to the variance within the model. The error term denotes the variation not accounted for in the model. Most of the variation occurred due to choice of method for RNA amplification and labeling. However, use of a volcano plot and heat map (Figures 2 and 3) were necessary to determine which kit provided superior data. 4766 out of 8732 genes meet the 0.05 FDR Significance criterion. |
Using a Volcano plot, which plots the log2
ratio between the MessageAmp II-Biotin Enhanced Kit
data and Kit A data vs. the Fisher’s least significant
difference pairwise comparisons method p value (Figure 2) [3],
it can clearly be seen that the difference between kits can be
attributed to increase in signal when using the MessageAmp II-Biotin Enhanced Kit.
Additionally a heat map (Figure 3) created using hierarchical
clustering to cluster both genes and methods shows the superior
performance of MessageAmp II-Biotin Enhanced Kit due
to higher signal. When each kit’s performance was measured
on a gene by gene basis, the average signal was increased for
91.3% of all genes when using the MessageAmp II-Biotin Enhanced Kit.
These results suggest that the aRNA generated by the MessageAmp
II-Biotin Enhanced Kit will lead to arrays with higher
signal intensity, allowing you to analyze more data points per
input sample than a leading competitor’s kit. The histogram
in Figure 4 provides another way to visualize the increase in
detectable genes at the low resolution end of the spectrum.
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Figure 2. Volcano Plot of MessageAmp™ II-Biotin Enhanced Kit vs Kit A. Plot of the log2 ratio between the MessageAmp™ II-Biotin Enhanced Kit data and Kit A data vs. the Fisher’s least significant difference pairwise comparisons method p value. (i.e., T test is performed just on differentially expressed data points, as identified by prior ANOVA analysis [3]). This plot shows higher signal intensity for data generated from the RNA amplified with the MessageAmp II-Biotin Enhanced Kit vs. Kit A. |
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Figure 3. Heat Map of Expression Values for MessageAmp II-Biotin Enhanced Kit and Kit A. Heat map comparing the four experimental conditions: MessageAmp™ II-Biotin Enhanced Kit vs. Kit A using a 4 and 14 hr IVT reaction. (Three arrays per condition are plotted via hierarchical clustering using Euclidean distance and average linkage.) Signal intensity is represented by color in a log2 scale from 1–14, where red is the highest and green is the lowest. There is very little difference between 4 and 14/16 hr IVT as evidenced in the heat map. |
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Figure 4. Histogram of Signal Distribution of All Array Features. An alternative presentation of the heat map data in Figure 3, distributions of signal intensities in histogram format illustrate that the Ambion MessageAmp™-II Biotin Enhanced Kit provides greater signal intensity (Median for Kit A 5.776 vs. Ambion 6.374). The dark shaded areas denote the samples at the lower end of the signal distribution (log transformed) and their increase between kits. Signal was increased for 91.3% of all genes. |
Arrays contain a huge number of data points.
Most of the interesting changes in expression are of low resolution,
and these are the ones scientists are most interested in investigating.
The data presented here shows that the MessageAmp II-Biotin Enhanced Kit
provides increased signal and increased Present calls, as well
as more data points at the low resolution end of the spectrum.
MessageAmp II-Biotin Enhanced Kit
The MessageAmp II-Biotin Enhanced Kit
includes enough reagents for the single-round amplification and
biotin-labeling of 20 samples. The kit has been shown to yield
sufficient aRNA for array analysis from as little as 50 ng input
total RNA with just a single round of amplification [4]. If your
experiments require two rounds of amplification, using the MessageAmp
II aRNA Amplification Kit for the first round of amplification
followed by the MessageAmp II-Biotin Enhanced Kit for
the second round with labeling is recommended.
Scientific Contributors
Charlie Johnson, Robert Setterquist, Sharmili
Moturi, Shikha Agarwal, Penn Whitley • Ambion, Inc.
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