Gene Expression Microarrays From Agilent

I started to use Agilent microarrays about three years ago. Now I am routinely running Agilent Gene Expression Whole Genome microarrays (human, mouse and rat). Agilent Whole Genome Expression microarrays contain more than 40000 unique genes and transcripts, all with public domain annotations. All features are referenced in the TIGR Resourcerer website (http://compbio.dfci.harvard.edu/tgi/). Over 70% of the represented probes are validated by Agilent's laboratory validation process. Traditionally, Agilent microarrays were two color microarrays. Nucleic acids from two different samples were labeled with two different dyes (usually Cy3 and Cy5), and the final result was the ratio of red and green signals, which represented the ratio of transcript copies in two samples. Recently Agilent introduced the One Color Gene Expression Platform.

Agilent microarrays are printed using 60-mer oligonucleotides on glass 1” x 3” slides. Such slides are common to all labs that print microarrays themselves, so all microarrays scanners that are suitable for homemade microarrays are suitable for Agilent microarrays.

Agilent microarrays are manufactured using a non-contact in situ synthesis process, base by base, from digital sequence files. This is done by using an inkjet process, which very accurately distributes extremely small volumes of chemicals to be spotted.

A novel experimental method for the selection and validation of highly specific oligonucleotide microarray probes has been developed by Agilent. This probe optimization approach is based on the supposition that, if multiple oligonucleotides for a particular gene are specific for that gene, then these probes will show very little difference in expression behavior. Optimized 60-mer oligonucleotides to represent genes are selected from groups of computationally determined candidate probes that show similar performance in several experiments for differential gene expression.

Agilent microarrays have very high specificity. I had an experience with transgenic mice overexpressing a human gene. Western blot and activity measurements showed a lot of this protein in the mice. However, the 60-mer oligonucleotide, printed on the Agilent Whole Mouse Genome Expression Microarray, had about 90% homology with the human gene. These microarrays did not hybridize with human transcript in multiple biological replicates.

I have used Agilent expression microarrays with different labeling protocols for nucleic acid. These microarrays work very well with directly or indirectly labeled cDNA and with directly or indirectly labeled antisense amplified RNA (aRNA). These microarrays worked very well even with the labeled RNA from paraffin embedded tissue.

My routine quality control criteria are: 1) Spots should not be flagged as “not found” or “absent”
2) For Whole Genome microarrays, the diameter of the spot has to be between 80 and 110 micron
3) Circularity of the spot has to be above 70%
4) More than 90% of pixels in the spot have to have signal intensity higher than background plus two standard deviations of background
5) Signal-to-noise ratio has to be 3 or above for each channel
6) Regression coefficient of the ratios for all pixels has to be 0.6 or above

Between 50% and 80% of spots of Agilent Gene Expression Microarrays routinely pass these rather stringent quality control criteria.

Agilent microarrays have incredible sensitivity. Agilent recommended_ hybridizing 750 ng of labeled nucleic acid from each sample for optimal results; the minimum of labeled material hybridized to microarray should be about 250 ng. However, I had an experience when I could hybridize only 120 ng of labeling material. The quality of this microarray was far from perfect, but the spots that passed quality control showed results in very good agreement with other replicates. Some experiments would be impossible with another microarray platform, which have lower sensitivity.

The only big disadvantage of Agilent gene expression microarrays, that I see, is the high cost of microarrays and processing of samples when using Agilent’s labeling kit.

As a conclusion, I highly recommend Agilent Gene Expression Microarrays, especially if one needs very high sensitivity and specificity.