From blank activated slides to labelling kits to instrumentation to analysis software, the technology developed by GE Healthcare can help you accelerate your microarray based research.

We also offer a free online tutorial that covers both microarray technology and the Codelink Bioarray platform.

The most common method is direct incorporation of fluorescent nucleotides into first-strand cDNA. The CyScribe First-Strand cDNA Labeling Kit utilizes this method. The second method is post labelling (amino allyl method). The CyScribe Post-Labeling Kit uses this method.

Both CyScribe kits have been designed to be used with dual-color microarray analysis in which differential expression of genes is being identified and quantified. Two different cDNA probes are prepared independently for this analysis; the first cDNA probe is transcribed using Cy3 as a label and the second probe using Cy5. Typically, one probe is prepared from control mRNA and the second from mRNA isolated from the treated cells or tissue under investigation. Both probes are hybridized together on the same microarray slide. The fluorescent signal from Cy3 and Cy5 labels can be determined. The relative intensities of the colored signals on individual spots are proportional to the amounts of specific transcripts in each sample, meaning differential gene expression can be examined. Careful normalization of fluorescence data either with internal spike standards or with house keeping genes is necessary for precise and reproducible results.

In order to acquire high quality data that will permit definitive conclusions about gene expression to be made, it is important that all steps in this procedure are optimized and standardized to reduce experimental error and variation. High quality labeling reagents are extremely important for generating highly fluorescent probes—CyDye fluorescent dyes combined with CyScribe Microarray Labeling Kits provide the solution for high sensitivity dual-color differential gene expression analysis.

Traditionally the probes have been labeled by direct incorporation of Cy3- or Cy5-labeled deoxynucleotides into first-strand cDNA during synthesis.

An alternative method gaining in popularity is post-labeling (amino allyl method). A chemically reactive nucleotide analog (amino allyl-dUTP) is easily incorporated into the cDNA and then subsequently labelled with the reactive forms of Cy3 or Cy5 dyes, which bind with equal efficiency to the cDNA. The amino allyl nucleotide is incorporated more efficiently into the cDNA than a bulky CyDye-labeled nucleotide resulting in probes more highly labelled with Cy3 or Cy5. Because the same amino allyl-dUTP is incorporated into both probes, the labeling efficiency is equivalent for both Cy3 and Cy5 reactive dyes.

Post labelling is a longer procedure than direct labeling as it includes two purification steps, however more cDNA is synthesized from the starting material as the synthesis of cDNA is not limiting and Cy3 and Cy5 are incorporated more evenly, the final yield of probes is increased in comparison to direct labeling.