Cisplatin-Digoxigenin mRNA Labeling for Nonradioactive Detection of mRNA Hybridized onto Nucleic Acid cDNA Arrays

T. Hoevel, H. Holz, M. Kubbies
1999 BioTechniques  
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 housekeeping gene expression but even superior on the detection limit of the expression of low abundant genes using
more » ... NA 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. Figure 2. Signal intensity profiles of housekeeping and low abundant genes applying nonradioactive mRNA hybridization. Panel A and B correspond to intensity profiles from Figure 1 indicated by the black and white arrow, respectively. Double peaks correspond to twin spots of identical genes. Panel A: p53 (a), nucleoside diphosphate kinase (b), GST theta-1 (c), ICH-2 protease (d), BCL-W (e), replication factor C (f) and GADD 153 (g). Panel B: CD40 L (a), microsomal GST (b), lymphotoxin beta (c), apoptotic cystein protease MCH4 (d), RAD (e), ERCC2 (f) and DNase X (g).
doi:10.2144/99275pf01 pmid:10572654 fatcat:s6hw5lpx5ffl7asc5qsu2ag4ju