RNA is often labeled with fluorescent dyes (i.e. Cy3 or Cy5) 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.

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