RNA is often labeled with fluorescent dyes, such as Alexa Fluor® 555 dye or Alexa Fluor® 647 dye (spectrally similar to Cy™3 or Cy™5), for microarray analysis. Label may be directly incorporated during cDNA synthesis, aRNA amplification or a post-aRNA reverse transcription (RT) reaction, or attached via a chemical coupling reaction performed after any enzymatic reactions (post-synthesis). Direct labeling requires the enzymatic incorporation of a Cy-labeled dNTP/NTP into the cDNA, aRNA or RT product. Alternatively, indirect labeling usually incorporates an intermediary nucleotide to which the fluorescent label is later attached. Although seemingly more convenient, direct labeling can be problematic for a number of reasons. Cy-labeled dNTPs/NTPs are not incorporated by reverse transcriptase or RNA polymerases with the same efficiency as unlabeled NTPs, leading to variable incorporation efficiency of different dyes (e.g. Cy3-NTP vs. Cy5-NTP). In addition, to be effective, direct incorporation often requires the use of more expensive modified nucleotides.

Post-synthesis labeling avoids the problems inherent to direct enzymatic incorporation of Cy-labeled dNTPs/NTPs. With indirect labeling, amine-modified NTPs (amino allyl-dUTP/UTP) are incorporated during reverse transcription or aRNA amplification. Amino allyl-NTPs are incorporated with similar efficiency as unmodified NTPs during enzymatic reactions such as reverse transcription. Coupling of Cy3 or Cy5 dyes to the amino allyl cDNA or aRNA, post-synthesis, results in probe populations with equivalent labeling efficiencies.

Amino allyl-dUTP contains a reactive amino group on a 2-carbon spacer attached to the methyl group on the base portion of dUTP. During the coupling reaction, after cDNA or aRNA synthesis, this amino group reacts with the NHS ester of a monoreactive Cy dye.

Ambion has several products for the post-synthesis labeling of nucleic acids for array analysis. The Amino Allyl cDNA Labeling Kit contains all the necessary reagents (except monoreactive Cy dyes) for the synthesis of amino allyl labeled cDNA by reverse transcription and subsequent Cy dye coupling. The MessageAmp™ aRNA Kit generates amplified aRNA for array analysis. Amino allyl UTP can be incorporated into the transcription step of this procedure to produce amino allyl labeled RNA ready for Cy dye coupling. Finally, amino allyl dUTP/UTP, available separately from Ambion, can be incorporated into any reverse transcription reaction.

Cy3 and Cy5 monoreactive dyes are available for Amersham Biosciences.
Cy3 and Cy5 are trademarks of Amersham Biosciences.