Ambion®'s Illumina® TotalPrep™ RNA Amplification Kit (patent pending) is a complete system for generating biotinylated, amplified RNA for hybridization with Illumina Sentrix® 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. 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.
The TotalPrep™ RNA Amplification Kit is based on the RNA amplification protocol developed in the laboratory of James Eberwine (Van Gelder et al. 1990). The procedure consists of reverse transcription with an oligo(dT) primer bearing a T7 promoter using Ambion’s 查看更多proprietary ArrayScript™, a reverse transcriptase (RT) engineered to produce higher yields of first strand cDNA than wild type enzymes. ArrayScript™ catalyzes the synthesis of virtually full-length cDNA, which is the best way to ensure production of reproducible microarray samples. The cDNA then undergoes second strand synthesis and clean-up to become a template for in vitro transcription with T7 RNA Polymerase. To maximize cRNA yield, Ambion’s proprietary MEGAscript® in vitro transcription (IVT) technology, along with biotin UTP (provided in the kit), is used to generate hundreds to thousands of biotinylated, antisense RNA copies of each mRNA in a sample
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