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Ambion® Amino Allyl cDNA Labeling Kit is designed for the two-step labeling of RNA to produce fluorescently labeled cDNA for use in glass array hybridizations. The nature of the reaction results in higher yield and much more efficiently labeled cDNA than direct incorporation. Sufficient reagents are included for 15 reactions.
• Amino allyl cDNA synthesis combined with secondary Cy™ dye coupling reaction results in consistent, uniform labeling of cDNA • Amino allyl cDNA synthesis is more efficient and less expensive than direct incorporation of Cy-NTPs
A Better Way to Incorporate Fluorescent Dyes Labeling cDNA for glass microarrays requires the incorporation of fluorescent dye into the cDNA. However, direct incorporation of dye-modified dNTPs by reverse transcriptase is inefficient, resulting in low yields and low specific activity cDNAs. Furthermore, there may be considerable variation in the incorporation efficiency of different dyes (e.g. Cy™3-dNTP vs. Cy™5-dNTP). The Amino Allyl cDNA Labeling Kit circumvents these problems by first incorporating a dNTP containing a reactive primary amine, then subsequently coupling it to the monoreactive ester form of the free dye. Incorporation of the amine-modified dNTP (amino allyl-dUTP) is as efficient as reverse transcription with unmodified dNTPs. A reactive amino group, on a 2-carbon spacer attached to the methyl group of dUTP, is available for the subsequent chemical reaction with the NHS ester form of any fluorophore (e.g. monoreactive Cy™3 and Cy™5 dyes). The efficiency of both the reverse transcription and coupling reactions result in greater yields, and in more uniformly and highly-labeled cDNA. This increases the sensitivity and reproducibility of array hybridizations. Because the Cy™ labeling is a secondary reaction dependent on amino allyl-dUTP incorporation, both Cy™3 and Cy™5 are coupled at the same rate resulting in equivalent labeling efficiencies.
Uniform Labeling Procedure First mRNA or total RNA is reverse transcribed incorporating amino allyl-modified dUTP into the synthesized cDNA. The template RNA is then degraded by base hydrolysis, and the reaction is neutralized. The amino allyl-modified cDNA is then purified to remove unincorporated nucleotides and primers. In the second step, the labeled cDNA is coupled with the monoreactive succinimide ester derivative of a Cy™ dye (not included in kit). The fluorescently labeled cDNA is purified with a NucAway™ Spin Column (included), to remove any unreacted dye. The resulting fluorescently labeled cDNA is ready for hybridization to glass arrays.
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