We use total RNA purified by standard procedures. Any commercial kit may be used. A list of kits used by us is shown here. A standard protocol is shown here. RNA should be treated with DNAse I and concentration determined photometrically. Normally, we require 5µg of RNA per array, however, we can also do microarray experiments with a lot less RNA if required.
RNA purification from tissues
For a protocol see here.
synthesis and labelling (low density microarrays)
At present, two commercially available kit systems are used. Initially a doublestranded cDNA is synthezised that is transcribed into a cRNA. Finally, during cRNA synthesis Cye3- or Cy5-conjugated nucleotides are either incorporated directly into the cRNA, or aminoallyl nucleotides are used during cRNA synthesis which are subsequently conjugated to Cy3 or Cy5 dyes. For a scheme of cRNA synthesis see here.
Hybridizations (low density
We routinely label all samples with the same dye (Cy3) and hybridize them onto individual microarrays. The advantage of this procedure is that dye-artefacts are excluded and that multiple comparison can be made across many different samples. We have also experienced that ratios are higher and more accurate if we hybridize cRNAs individually onto the Inflammation array. The prerequisiste for this strategy is a highly standardized experimental procedure. Ratios of gene expression comparing two samples are obtained virtually using Excel or CytoBASE (for an example view here).
Standard protocol for “single channel” DNA-Microarray hybridizations pdf 239kB (low density microarrays)
For high density microarray experiments, reverse transcription-,
amplification-, fluorophore labeling-, cRNA fragmentation- and
hybridization-procedures are all performed using Agilent chemistry and
highly standardized work flows (for a brief description see here)