TargetAmp™ aRNA Amplification Performance

TargetAmp™: Selection Guide | Process | Performance | FAQ | Poster (3 MB PDF)

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.

HeLa Total RNA HeLa Poly(A) RNA
(assume 2% of total RNA is Poly(A) RNA)
Aminoallyl-aRNA Yield Fold-Amplification of Poly(A) RNA
25 ng 0.5 ng 2.9 μg 5800
50 ng 1.0 ng 5.2 μg 5200
100 ng 2 ng 11.4 μg 5700
500 ng 10 ng 69.9 μg 6990

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.

Amount of Total RNA Amplified aRNA yield from Total Rat Brain RNA
(Poly(A) RNA 5% of total rat brain RNA)
aRNA yield from Total HeLa RNA
(Poly(A) RNA 2% of total HeLa RNA)
aRNA yield from Total Rat Kidney RNA
(Poly(A) RNA 1.5%-2% of total rat kidney RNA)
10 pg (1 cell) 3 µg (6 x 106) 1.9 µg (9.5 x 106) 1.4 µg (9.3 x 106)
50 pg (5 cells) 21 µg (8.4 x 106) 9 µg (9 x 106) 7 µg (9.3 x 106)
100 pg (10 cells) 54 µg (10.8 x 106) 19 µg (9.5 x 106) 17 µg (11 x 106)
200 pg (20 cells) 75 µg (7.5 x 106) 37 µg (9.2 x 106) 33 µg (11 x 106)
500 pg (50 cells) 143 µg (5.7 x 106) 74 µg (7.4 x 106) 71 µg (9.4 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 (SYBR� 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.

Fold Amplification 3’/5’ ratio GAPDH 3’/5’ ratio β-actin % Present Calls Reproducibility (2 amplifications)
>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.

Fold Amplification of Poly(A) RNA Average 3’/5’ ratio GAPDH Average 3’/5’ ratio β-actin Average Present Calls Reproducibility (2 amplifications)
>5 x 106 5.6 +/- 1.5 10.7 +/- 0.07 50.7 +/- 1% 0.99