Preparing Fluorescent Probes for Microarray Studies

A number of articles have been published describing methods to produce fluorescent probes from RNA (or DNA) samples. These methods are conceptually similar. Broadly speaking, they involve some or all of the following procedures: template amplification, template transcription with concomitant incorporation of modified bases into the transcribed products (DNA or RNA), and signal amplification. The simplest technique relies on the direct incorporation of Cy3- or Cy5-labeled nucleotides into oligo-dT-primed cDNA by reverse transcriptase (RT). This method has proven to be reasonably robust, but it requires 50–200 μg of total RNA or 2–5 μg of poly(A) RNA per labeling (1 –4 ). Thus, rather large tissue samples are required for each study.