Cisplatin-digoxigenin mRNA labeling for nonradioactive detection of mRNA hybridized onto nucleic acid cDNA arrays.
We optimized a novel nonradioactive hybridization technique using a cis-platin coupled digoxigenin derivative for direct labeling of mRNA. This new mRNA reporter molecule was applied to cDNA membrane arrays to simultaneously identify expression of hundreds of genes. The sensitivity of this nonradioactive mRNA hybridization technique was comparable to radioactive cDNA labeling on house-keeping gene expression but even superior on the detection limit of the expression of low abundant genes using mRNA isolated from human diploid fibroblasts. Additional advantages are faster readout and decreased total working times because of luminescence technology and avoidance of radioactivity. Finally, no potential artifactual reverse transcription step is necessary because of direct labeling of mRNA used for hybridization on nucleic acid arrays.
Hoevel
T
T
Roche Pharmaceutical Research, Dept. Cell Analytics, Penzberg, Germany. thorsten.hoevel@roche.com
Holz
H
H
Kubbies
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M
D002945:Cisplatin / Q000737:chemistry*; D018076:DNA, Complementary / Q000235:genetics*; D004076:Digoxigenin / Q000737:chemistry*; D005347:Fibroblasts; D015870:Gene Expression; D006801:Humans; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012333:RNA, Messenger; D004076:Digoxigenin; D002945:Cisplatin