Fluorescent labelling of cRNA for microarray applications

Microarrays of oligonucleotide expression libraries can be hybridised with either cDNA, generated from mRNA during reverse transcription, or cRNA, generated in an Eberwine mRNA amplification procedure. While methods for fluorescent labelling of cDNA have been thoroughly investigated, methods for cRNA labelling have not. To this purpose, we developed an aminoallyl‐UTP (aa‐UTP) driven cRNA labelling protocol and compared it in expression profiling studies using spotted 7.5 K 65mer murine oligonucleotide arrays with labelling via direct incorporation of Cy‐UTPs. The presence of dimethylsulfoxide during coupling of aa‐modified cRNA with N‐hydroxysuccinimide‐modified, fluorescent Cy dyes greatly enhanced the labelling efficiency, as analysed by spectrophotometry and fluorescent hybridisation signals. Indirect labelling using aa‐UTP resulted in 2‐ to 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 nt). Incorporation of more label increased Cy3 signal but lowered Cy5 fluorescence. This effect is probably due to quenching, which is more prominent for Cy5 than for Cy3. In conclusion, the currently developed method is an efficient, robust and inexpensive technique for fluorescent labelling of cRNA and allows sensitive detection of gene expression profiles on oligonucleotide microarrays.