Table 2 Synthesis and hybridization of fluorescently labeled cDNA derived from sample RNA

Hybridization of the microarray with sample representing the mRNA population is accomplished by synthesizing complementary DNA (cDNA) using reverse transcriptase. Microarray experiments in prokaryotic organisms cannot utilize the poly(A) tails found in eukaryotic mRNA; thus, alternatives to the highly successful methods employed for generating fluorescent cDNA using oligo-dT to prime the reverse transcription labeling reaction have to be employed.

   • cDNA synthesis Tests comparing random hexamers and random octamers gave essentially indistinguishable results. On the other hand, critical parameters for maximizing signal-to-noise were: 1.) high level of incorporation of fluorescent dye by using a high ratio of amino allyl dUTP in the reverse transcription reaction and saturating amount of reactive dye during the coupling of activated dye; and 2.) the use of two purification steps relying on independent physical separation principals applied to the test sample: first after cDNA synthesis and second, after the coupling of fluorescent dye. Direct chemical labeling of RNA, while attractive, did not allow as high signal-to-noise during our tests. Similarly, pilot experiments using pooled 3' gene-specific oligos as cDNA primers did not give better results.

   • Hybridization: While increases in signal-to-noise were associated with optimization of fluorescent sample production (above), a fairly wide range of standard microarray pre-hybridization and hybridization protocols were found to give comparable results. However, critical aspects of the described method were keeping the microarray slide warm during the addition of sample to the surface following pre-hybridization and ensuring that the slide remains hydrated prior to the final drying immediately before scanning.