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aRNA, Aminoallyl-aRNA, or Biotin-aRNA
Yield and Fold-Amplification
The TargetAmp RNA Amplification Kits are optimized for use with SuperScript
II and SuperScript III Reverse Transcriptase enzymes and incorporate EPICENTRE
Biotechnologies' AmpliScribe™ T7 High Yield in vitro transcription
technology. In addition, the kits utilize a proprietary chemistry that virtually
eliminates non-specific amplification products. The result is a TargetAmp RNA
amplification reaction that produces extremely high yields of aRNA (or aminoallyl-aRNA
or biotin-aRNA) with virtually no non-specific amplification product.
Table 1. Yields of aRNA obtained using the TargetAmp™ 1-Round
aRNA Amplification Kit 103. Results are the average of multiple experiments
using the HeLa Total RNA. Yields of aminoallyl-aRNA using the TargetAmp™ 1-Round
aRNA Amplification Kit 101 and biotin-aRNA using the TargetAmp™ 1-Round
Biotin-aRNA Amplification Kit 105 are essentially the same.
| 25 ng |
0.5 ng |
2.9 μg |
5800 |
| 100 ng |
2 ng |
11.4 μg |
5700 |
Table 2. Yields of aRNA obtained using the TargetAmp™ 2-Round
aRNA Amplification Kit 2.0.
Results are the average of multiple experiments using total RNA from three
sources, each with a different Poly(A) RNA content. The fold-amplification of
the Poly(A) RNA contained in each is shown in parentheses. Less than 1 µg
of non-specific amplification product was produced in the “no-RNA” control
reaction. Yields of aminoallyl-aRNA produced by the TargetAmp™ 2-Round
Aminoallyl-aRNA Amplification Kit 1.0 are essentially the same.
| 50 pg (5 cells) |
21 µg (8.4 x 106) |
9 µg (9 x 106) |
7 µg (9.3 x 106) |
| 200 pg (20 cells) |
75 µg (7.5 x 106) |
37 µg (9.2 x 106) |
33 µg (11 x 106) |
Size of the aRNA Produced
The TargetAmp 1-Round Aminoallyl-aRNA Kit 101, the TargetAmp 1-Round aRNA Amplification
Kit 103, and the TargetAmp™ 1-Round Biotin-aRNA Amplification Kit 105
produce aminoallyl-aRNA, unlabeled aRNA, and biotin-aRNA respectively with
an average size of about 1200 bases as analyzed by both denaturing agarose
gel electrophoresis and the Agilent 2100 Bioanalyzer.
The size of the aminoallyl-aRNA produced by the TargetAmp 2-Round Aminoallyl-aRNA
Kit 1.0 was investigated using two different amounts of input total RNA. Total
RNA from a single laser captured rat dorsal root ganglion cell (about 10 pg)
and 200 pg of total RNA from rat brain were independently amplified using the
TargetAmp 2-Round Aminoallyl-aRNA Amplification Kit 1.0. The size distribution
of the resulting aminoallyl-aRNA produced from the rat dorsal root ganglion
cell was analyzed by denaturing agarose gel electrophoresis (Figure 1a) and
the aminoallyl-aRNA generated from the rat brain cells was analyzed using an
Agilent 2100 Bioanalyzer (Figure 1b). In both examples, the aminoallyl-aRNA
produced had an average size of greater than 600 bases.
A
 |
B
 |
Figure 1. The TargetAmp™ 2-Round Aminoallyl-aRNA Amplification
Kit 1.0 produces AA-aRNA with a size distribution between 200 to 4000 bases.
A. Denaturing agarose gel analysis of AA-aRNA produced from 2 independent
single-cell RNA amplification reactions.
B. Agilent 2100 Bioanalyzer analysis of AA-aRNA produced from 200 pg
of rat brain total RNA. |
Reproducibility of the TargetAmp
RNA Amplification Reaction
Reproducible RNA amplification results are critical to the success of long-term
gene expression studies using DNA microarrays. The RNA amplification reproducibility
of the TargetAmp 2-Round Aminoallyl-aRNA Kit 1.0 was demonstrated under the
most extreme conditions - amplifying total RNA from a single laser-captured
cell. Two samples of total cellular RNA, each isolated from one laser captured
cell from rat dorsal root ganglion section, were amplified independently using
the TargetAmp 2-Round Aminoallyl-aRNA Amplification Kit 1.0. The resulting aminoallyl-aRNAs
were labeled with Cy3-NHS and hybridized in parallel to rat arrays. A scatter
plot of the relative fluorescent intensities of the genes detected on the two
arrays is shown below. The correlation coefficient was 0.9932 (R2=0.9932)
indicating highly reproducible amplification.
 |
Figure 2. Two independent RNA amplification reactions, each using total
RNA from a single laser captured rat dorsal root ganglion cell, amplified
using the TargetAmp™ 2-Round Aminoallyl-aRNA Amplification Kit 1.0,
and then labeled with Cy3-NHS, produced a correlation coefficient of 0.9932,
indicative of highly reproducible RNA amplification. |
Preservation of Gene Expression Levels
(Preservation of Relative Abundance of Transcripts)
The relative expression level of a panel of genes in Universal Human Reference
RNA (Stratagene) and adult skeletal muscle total RNA was assessed before and
after TargetAmp 1-Round and TargetAmp 2-Round amplification reactions. Unamplified
RNA and aRNA produced by a TargetAmp 1-Round and a TargetAmp 2-Round amplification
reaction was reverse transcribed using random primers and Superscript III Reverse
Transcriptase. Quantitative PCR (SYBRŪ Green detection) was performed using
EPICENTRE Biotechnologies' TAQurate GREEN Real-Time PCR MasterMix. Beta-2 microglobulin
was used for expression level normalization. The differences in expression levels
between the RNA samples are represented as the normalized ΔCT in
qPCR cycles (ΔΔCT). The results presented in the chart
below demonstrate that both the TargetAmp 1-Round and 2-Round amplification
reactions preserve the relative expression levels of genes.
Correlation of Differential
Gene Expression (Fidelity of Amplification)
To assess the correlation of differential gene expression biotin-aRNA was prepared
from total RNA of 6-day and 12-day cultured mouse neural cells using the TargetAmp
1-Round Aminoallyl-aRNA Amplification Kit 101, TargetAmp 2-Round Aminoallyl-aRNA
Amplification Kit 1.0 and the standard Affymetrix biotin-aRNA labeling procedure.
Total RNA from 6-day and 12-day mouse neural cells was amplified independently
using the TargetAmp 1-Round Kit 101 procedure and 400 pg of total RNA was amplified
using the TargetAmp 2-Round Aminoallyl-aRNA Amplification Kit 1.0 The aminoallyl-aRNA
produced by the 2 kits was then labeled with Biotin-X-X-NHS by standard procedure.
Approximately 5 µg of total RNA was biotin-labeled using the standard
Affymetrix labeling procedure.
The resulting biotin-aRNA samples were fragmented and hybridized to Affymetrix
GeneChip Mouse Genome 430 2.0 Arrays, in triplicate. Differentially expressed
genes were identified using the GeneSpring software package (Silicon Genetics).
The expression ratios obtained with the Affymetrix protocol and with the TargetAmp
1-Round Aminoallyl-aRNA Amplification Kit 101 protocol are plotted in Figure
3A below. The correlation coefficient of 0.90 demonstrates good agreement between
differentially expressed genes identified by the TargetAmp Kit 101 protocol
and by the standard Affymetrix labeling method.
The expression ratios obtained with the TargetAmp 2-Round Aminoallyl-aRNA
Kit 1.0 protocol and with the Affymetrix protocol are plotted in Figure 3B.
A correlation coefficient of 0.89 demonstrates good agreement between differentially
expressed genes identified using biotin-aRNA target produced by the TargetAmp
Kit 1.0 and conjugated to Biotin-X-X-NHS and by the standard Affymetrix labeling
method.
 |
Figure 3A. Differentially expressed genes identified using biotin-aRNA
produced by the TargetAmp™ 1-Round Aminoallyl-aRNA Amplification Kit
101, and subsequent biotinylation, and biotin-aRNA produced by the standard
Affymetrix T7 RNA polymerase protocol gave a correlation coefficient of 0.90. The
'Y' axis shows 12-day vs. 6-day gene expression ratios obtained using biotin-aRNA
target produced by the TargetAmp 1-Round Kit 101 protocol. The 'X' axis shows
the 12-day vs. 6-day gene expression ratios obtained using biotin-aRNA target
produced by the Affymetrix standard procedure. |
 |
Figure 3B. Correlation of differentially expressed genes identified
by the TargetAmp™ 2-Round Aminoallyl-aRNA Amplification Kit 1.0 protocol
and the standard Affymetrix biotin-aRNA labeling procedure. The 'Y' axis
shows 12-day vs. 6-day cultured mouse neural cell gene expression ratios
obtained with biotin-aRNA target produced by the TargetAmp 2-Round Kit 1.0
protocol. The 'X' axis shows the 12-day vs. 6-day gene expression ratios
obtained with biotin-aRNA target produced by the Affymetrix standard procedure. |
Quality Assessment on GeneChip® Arrays (3'
to 5' Ratios and % Present Calls)
The average of the 3’-to-5’ ratios and % Present Calls obtained
using biotin-aRNA produced by the TargetAmp 1-Round Aminoallyl-aRNA Amplification
Kit 101 and Biotin-X-X-NHS are shown in Table 3. These values are well within
the quality assessment guidelines established by Affymetrix for target biotin-aRNA
performance, and demonstrate that the TargetAmp Kit 101 yields high quality
expression profile data using Affymetrix GeneChip Arrays.
Table 3. AA-aRNA produced using the TargetAmp™ 1-Round Aminoallyl-aRNA
Amplification Kit 101, and subsequently biotinylated, produced high quality
GeneChip Arrays as determined by Affymetrix quality assessment guidelines.
| >5000 |
1.05 +/- 0.05 |
2.8 +/- 0.01 |
56.3 +/- 2% |
0.99 |
Performance of biotin-aRNA prepared using the TargetAmp 2-Round Aminoallyl-aRNA
Amplification 1.0 protocol was also evaluated on the Affymetrix GeneChip
platform. Four hundred picograms (400 pg) of total RNA from 6-day and 12-day
cultured mouse neural cells was independently amplified using the TargetAmp
2-Round Aminoallyl-aRNA Amplification Kit 1.0. The resulting aminoallyl-aRNA
was labeled by conjugation to EPICENTRE's Biotin-X-X-NHS using a standard conjugation
procedure. The resulting biotin-aRNA was fragmented and hybridized to GeneChip
Mouse Genome 430 2.0 Arrays, in triplicate, by the Gene Expression Center of
the University of Wisconsin Biotechnology Center. The averages of the 3’-to-5’ ratios
and Present Calls were determined with the MAS 5 software (Affymetrix) and
are shown in Table 4. The data demonstrates the high quality arrays that are
obtained with biotin-aRNA target prepared using the TargetAmp 2-Round Aminoallyl-aRNA
Amplification Kit 1.0 and Biotin-X-X-NHS.
Table 4. AA-aRNA produced using the TargetAmp™ 2-Round Aminoallyl-aRNA
Amplification Kit 1.0, and subsequently biotinylated, generated high quality
GeneChip Arrays.
| >5 x 106 |
5.6 +/- 1.5 |
10.7 +/- 0.07 |
50.7 +/- 1% |
0.99 |
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