Gene Expression Analysis |
RNA Amplification
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Applications
Production of sense RNA (sRNA) from 10-500 ng of total RNA. Use the amplified
sRNA for:
- Archiving valuable RNA samples for future use.
- Microarray studies requiring either sRNA target or labeled antisense cDNA
target for improved 5´/3´ coverage.
- Real-time PCR studies of gene expression.
Or, convert the amplified sRNA to dsDNA using the dsDNA Conversion Kit for
RiboMultiplier™ for:
- Target for Roche-NimbleGen expression arrays.
- Preparation of templates for high-throughput (next-gen) sequencing.
- cDNA library construction.
The RiboMultiplier™ Sense-RNA Amplification Kit* generates amplified
sense-strand RNA (sRNA) from the mRNA contained in a total RNA sample (Fig.
1). First-strand cDNA is synthesized from total RNA, and the RNA is hydrolyzed.
Next, an oligo-nucleotide containing a terminal-tagging sequence (TTS), a 3´-blocked
terminus, and a random sequence is used to generate cDNA molecules with a known
sequence tag at their 3´ ends. A double-stranded T7 promoter sequence
is then added to the 3´ ends of the cDNA molecules by DNA synthesis, followed
by in vitro transcription, which results in greater than 10,000-fold
amplification of the original message. The entire sRNA procedure is performed
in a single reaction tube with a hands-on time of less than 1 hour.
Benefits
- sRNA shows excellent correlation with unamplified samples, indicating
a faithful representation of the original gene expression profile (Fig.
2).
- The unique terminal-tagging procedure yields sRNA with an excellent 3´/5´ ratio
and less 3´ bias compared to other oligo(dT)-primed RNA amplification
processes (Table 1).
- Microgram amounts of sRNA are generated from as little as 10 ng of input
total RNA (Table 2), with essentially no background amplification.
- The entire RiboMultiplier sRNA kit procedure can be completed in 1 day.
|
Figure 1 (click to enlarge). Schematic overview of the RiboMultiplier
Sense-RNA Amplification procedure. |
A |
B
 |
Figure 2 (click to enlarge). Correlation of differential gene expression
data from sRNA produced by the RiboMultiplier™ Sense-RNA Amplification
Kit and unamplified total RNA with MAQC TaqMan® assays (~1,000
genes). Plots of log2 gene expression ratios for human brain
and universal human reference total RNA obtained for ds cDNA synthesized
from either RiboMultiplier sRNA (Panel A, X-axis) or unamplified total RNA
(Panel B, X-axis) versus corresponding known MAQC TaqMan ratios (Y-axis)
showed a similarly high correlation value (r=0.92), demonstrating excellent
fidelity of the amplified sRNA compared to the unamplified total RNA. |
| GUSB |
2,245 |
0.8 |
36.8 |
| TUBA |
1,706 |
0.9 |
223 |
| ENSA |
2,512 |
0.9 |
24.3 |
| ACTB |
1,792 |
0.3 |
73.5 |
| TFRC |
5,010 |
0.3 |
256 |
| GAPDH |
1,310 |
1.2 |
181 |
| PKG1 |
2,338 |
0.4 |
24.3 |
| H3F3A |
1,117 |
1.6 |
121 |
| ALB |
2,215 |
1.5 |
9.8 |
| HPRT |
1,331 |
0.3 |
6.1 |
|
Table 1. 3´/5´ ratios for RiboMultiplier™ sRNA compared
to aRNA produced by another RNA amplification procedure. Random-primed
cDNA samples prepared from either RiboMultiplier™ sRNA or T7-dT aRNA
were used in qPCR, and the 3´/5´ sequence ratios were determined
for several genes. In general, 3´/5´ ratios around 1.0 were seen
for the RiboMultiplier sRNA sample, demonstrating the enhanced 5´ sequence
representation in sRNA as compared to T7-dT aRNA. |
10 ng
|
8.2 µg |
7.3 µg |
| 25 ng |
23.4 µg |
17.5 µg |
| 100 ng |
86.7 µg |
71.8 µg |
|
Table 2. The RiboMultiplier™ sRNA Kit provides high yields
of sRNA from as little as 10 ng of total RNA.
a. Approximately 2% of HeLa total RNA is poly(A) RNA. |
*Patent pending.
MasterPure™ Complete DNA and RNA Purification Kit
MessageBOOSTER™ cDNA Synthesis Kit for qPCR
MessageBOOSTER™ Whole-Transcriptome cDNA Synthesis Kit for qPCR
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