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Improving RNA Amplification

based aRNA amplification kit. The MessageAmp II cRNA Amplification Kit incorporates extensive improvements to the original MessageAmp procedure. In developing MessageAmp II, Ambions scientists focused on the reverse transcription and second strand cDNA synthesis steps, both of which are critical for generating high yields of full-length labeled cRNA. This led to the development of ArrayScript, an engineered M-MLV reverse transcriptase, and to the optimization of the second strand cDNA synthesis reaction to synergize with the improvements gained with ArrayScript. One of the principal benefits conferred by ArrayScript and the optimized second-strand synthesis step is that MessageAmp II will generate sufficient labeled cRNA in a single round of amplification for array analysis from as little as 100 ng of total RNA (Figure 1). Other commercially available products for cRNA amplification usually demand two rounds of amplification at this level of input.

Figure 1. High Density Oligonucleotide Microarray Hybridized with MessageAmp II cRNA. Shown is a portion of a GeneChip array image used in this report. All RNA samples were amplified and labeled with biotin-16-UTP/-11-CTP (PerkinElmer Life Sciences), with the MessageAmp II Kit. Biotin labeled cRNA was fragmented, then hybridized and stained using GeneChip Human Genome Focus Arrays following the GeneChip Expression Analysis technical manual.


Together, the RT and second strand cDNA reactions result in an increase in the conversion of mRNA into longer double-stranded cDNA templates. This increase allows both shortened in vitro transcription (IVT) reaction incu
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