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.

[1]  P. Warren,et al.  Gene expression microarrays and the integration of biological knowledge. , 2001, Trends in biotechnology.

[2]  Angela Relógio,et al.  Optimization of oligonucleotide-based DNA microarrays. , 2002, Nucleic acids research.

[3]  Yidong Chen,et al.  Amine-modified random primers to label probes for DNA microarrays. , 2002 .

[4]  C Schwager,et al.  Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays. , 2002, BioTechniques.

[5]  E. Brown,et al.  Quantitative analysis of mRNA amplification by in vitro transcription. , 2001, Nucleic acids research.

[6]  Neil Winegarden,et al.  Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA , 2002, Nature Biotechnology.

[7]  C. Riener,et al.  Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin. , 2000, Bioconjugate chemistry.

[8]  P. Brown,et al.  Exploring the metabolic and genetic control of gene expression on a genomic scale. , 1997, Science.

[9]  Johan T den Dunnen,et al.  A common reference for cDNA microarray hybridizations. , 2002, Nucleic acids research.

[10]  Jiasen Lu,et al.  Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays. , 2000, Nucleic acids research.

[11]  E. Winzeler,et al.  Genomics, gene expression and DNA arrays , 2000, Nature.

[12]  Christian A. Rees,et al.  Molecular portraits of human breast tumours , 2000, Nature.

[13]  N. Yamamoto,et al.  Microarray fabrication with covalent attachment of DNA using Bubble Jet technology , 2000, Nature Biotechnology.

[14]  Yudong D. He,et al.  Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer , 2001, Nature Biotechnology.

[15]  Shigeo Yoshida,et al.  Evaluation and optimization of procedures for target labeling and hybridization of cDNA microarrays. , 2002, Molecular vision.

[16]  F. Marincola,et al.  High-fidelity mRNA amplification for gene profiling , 2000, Nature Biotechnology.

[17]  B. S. Baker,et al.  Gene Expression During the Life Cycle of Drosophila melanogaster , 2002, Science.

[18]  J. Eberwine,et al.  Amplified RNA synthesized from limited quantities of heterogeneous cDNA. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[19]  R. Stoughton,et al.  Experimental annotation of the human genome using microarray technology , 2001, Nature.