• For the synthesis and purification of fluorescently labeled cDNA for glass array analysis
  
  
• 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
  
  

The 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.

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. Cy3-dNTP vs. Cy5-dNTP). The Amino Allyl cDNA Labeling Kit circumvents these problems by first incorporating a dNTP containing a reactive primary amine, is subsequently coupled 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 Cy3 and Cy5 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 Cy3 and Cy5 are coupled at the same rate resulting in equivalent labeling efficiencies.

The figure at left illustrates the similar labeling efficiencies of the Cy3 and Cy5 dyes when the Amino Allyl cDNA Labeling Kit is used. Reverse transcription was performed using 5 µg of poly(A) RNA. The reaction was split and each aliquot was coupled with either Cy3 or Cy5 monoreactive dyes. Both labeled cDNAs were then hybridized to a single array and data was collected with the Gene Pix 4000 Microarray Scanner (Axon Instruments, Inc.). The resulting scatterplot shows little difference between the hybridization pattern of the Cy3 and Cy5 labeled cDNA.

Uniform Labeling Procedure
The schematic at left illustrates the Amino Allyl cDNA Labeling Kit 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 (available from Amersham Pharmacia Biotech). 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.

A Complete Kit for Synthesizing Amino Allyl cDNA
The Amino Allyl cDNA Labeling Kit contains reverse transcription reagents, oligo(dT) and random primers, RNA hydrolysis and neutralization reagents, Coupling Buffer, and NucAway Spin Columns for sample clean-up. Also included are reagents for solubilization of the dye esters, reagents for reaction quenching, controls and a detailed Instruction Manual. Monoreactive Cy dyes are not included.

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