TargetAmp™ 2-Round Aminoallyl-aRNA Amplification Kit 1.0

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Note: The procedure for this TargetAmp Kit has changed. Please download the latest version of the kit protocol before proceeding.

Applications

  • Amplification and aminoallyl-labeling of mRNA in total RNA of 1-50 cells (10-500 pg RNA) for use in microarray analysis. Ideal for small samples such as laser-capture microdissected cells, biopsy samples, cultured cells, or other minute cell or tissue samples.

The TargetAmp™ 2-Round Aminoallyl-aRNA Amplification Kit 1.0* generates at least 5-million-fold amplification of the poly(A) RNA contained in a total cellular RNA sample. This amplification is sufficient to generate microgram amounts of aminoallyl-labeled antisense RNA (AA-aRNA; also called AA-cRNA) from the total RNA contained in a single cell. The kit uses an Eberwine-type linear RNA amplification method,1 but incorporates a number of improvements that significantly increase the yields of AA-aRNA obtained, while reducing the level of nonspecific amplification products. The kit is recommended for amplifying mRNA in total RNA from 1 to ~50 cells.

The kit provides all reagents (except reverse transcriptases and RNA purification columns) for generating AA-aRNA. The entire procedure takes only ~4 hours of hands-on time and can be completed in 2 days. Multiple samples can be amplified simultaneously. Once produced, the aminoallyl-aRNA can be readily labeled using biotin-NHS or fluorescent dye-NHS compounds. Biotin-NHS is available from Epicentre.

Benefits

  • The kit generates microgram amounts of AA-aRNA from as little as 10 pg of total cellular RNA (Table 1 and Fig. 1), representing greater than 5,000,000-fold amplification.
  • The entire two-round RNA amplification process can be completed in 2 days and requires only about 4 hours of hands-on time.
  • Multiple RNA samples can be easily amplified simultaneously.
  • Highly reproducible amplification and microarray results.

References

  1. Van Gelder, R.N. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1663.
A  
 
B C
Figure 1. The TargetAmp™ Kit 1.0 amplifies RNA from a single cell. A: Laser capture of a single cell from a section of rat dorsal root ganglion (DRG). B: Agarose gel of the ~1-3 µg AA-aRNA obtained using the TargetAmp Kit 1.0 to amplify mRNA in the ~10 pg total RNA isolated from two different laser-captured DRG cells. C: Scatter plot of microarray results obtained when Cy™ dye-labeled aRNA target prepared using the TargetAmp Kit 1.0 was hybridized to a glass slide spotted with rat brain cDNA.
Amount
of Input
Total RNA		 AA-aRNA Yield
from Rat Brain
Total RNAa		 AA-aRNA Yield
from HeLa
Total RNAb		 AA-aRNA Yield
from Rat Kidney
Total RNAc
10 pg 3 µg 1.3 µg 1.4 µg
50 pg 19 µg 8 µg 7 µg
100 pg 53 µg 19 µg 15 µg
500 pg 161 µg 71 µg 72 µg

Table 1. The TargetAmp™ Kit 1.0 produces microgram amounts of AA-aRNA from as little as 10 pg (one cell) of total RNA. Results represent the average of multiple experiments.

 

a. Approximately 5% of rat brain total RNA is poly(A) RNA.
b. Approximately 2% of HeLa total RNA is poly(A) RNA.
c. Approximately 1.5%-2% of rat kidney total RNA is poly(A) RNA.

*TargetAmp™ aRNA Amplification Products are covered by intellectual property licensed to Epicentre Technologies Corporation from Johnson & Johnson Pharmaceutical Research & Development, L.L.C., and by intellectual property sublicensed to Epicentre Technologies Corporation from Incyte Corporation.