Peter A.C. 't Hoen1, Floor de Kort1,3,4, Gert-Jan B. van Ommen1, Johan T. den Dunnen1,2
1Center for Human and Clinical Genetics, and 2Leiden Genome Technology Center, Leiden Univeristy Medical Center and 3Pamgene B.V., Den Bosch 4Present address: Department of Gastroenterology, Amsterdam Medical Center E-mail: p.a.c.hoen @ lumc.nl
Expression profiling studies on microarrays generally require considerable amounts of input RNA (20-50 mg of total RNA). When the RNA is amplified before application on microarrays, the amount of starting material can be highly reduced. To further enhance sensitivity limits, we developed a new and robust protocol for fluorescent labelling of amplified RNA (cRNA). During linear amplification, aminoallyl-UTP (aa-UTP) is incorporated into the cRNA, which is subsequently coupled with fluorescent Cy-dyes. The presence of dimethylsulfoxide during coupling greatly enhanced the labelling efficiency, as analysed by spectrophotometry, and fluorescent hybridisation signals. Indirect labelling using aa-UTP resulted in 2-3-fold higher degrees of labelling and fluorescent signals than labelling by direct incorporation of Cy-UTP. By variation of the aa-UTP:UTP ratio, a clear optimal degree of labelling was found (1 dye per 20-25 nucleotides). Incorporation of more label increased Cy3 signals but lowered Cy5 fluorescence. This effect is probably due to quenching, which is more prominent for Cy5 than for Cy3. The procedure, in which linear amplification is combined with high fluorescent labelling efficiency, thus allows for reliable determination of gene expression profiles in samples from which little RNA is recovered.