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Illumina® TotalPrep™ RNA
Amplification Kit
Amplify and Biotinylate RNA for Use on Illumina® Sentrix® Arrays
• Perform amplification from just
50 ng input RNA
• Developed for expression profiling using Sentrix® BeadChips
• Bio-16-UTP included in optimized NTP mix, reducing set up time and maximizing
labeling
The Illumina® TotalPrep™ RNA Amplification
Kit (patent pending) is a complete system for generating biotinylated,
amplified RNA for hybridization with Illumina Sentrix arrays.
RNA amplification has become the standard method
for preparing RNA samples for array analysis [1–2]. The Illumina
RNA Amplification Kit was developed for use with Illumina Sentrix
arrays. It is based on the RNA amplification protocol developed
in the laboratory of Dr. James Eberwine [3]. The procedure consists
of reverse transcription with an oligo(dT) primer bearing a T7
promoter and in vitro transcription of the resulting cDNA with
T7 RNA Polymerase to generate hundreds to thousands of antisense
RNA copies of each mRNA in a sample. The Illumina RNA Amplification
procedure maintains a close concordance to the basic Eberwine
protocol. The labeled cRNA produced with the kit is ready for
hybridization with Illumina arrays.
The new TotalPrep RNA Amplification Kit incorporates
extensive improvements to the in vitro transcription reaction
(IVT) over the previous Illumina RNA Amplification Kit. The IVT
reaction has been optimized for the most effective biotin labeling,
and an NTP mix containing biotinylated UTP has been added to
the kit, allowing ease of use and optimal labeling. These modifications
to the kit have significantly increased sensitivity, providing
increased Percent Present calls and single round amplification
with just 50 ng of input RNA. Figure 1 demonstrates the amplified
RNA yields possible from inputs ranging from 10–100 ng from two
different sources.
The kit contains reagents for 24 single-round
amplification reactions.
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| Figure
1. cRNA Yield with Different Amounts of
Input RNA. The indicated amounts of Control RNA
(HeLa total RNA) or Stratagene Human Universal Reference
RNA were amplified in triplicate using a 14 hr IVT incubation
times (average yield shown). |
Scientific Contributors
Robert Setterquist, Sharmili Moturi • Ambion,
Inc. |
