QUANTITATIVE AND SENSITIVE NORTHERN BLOT HYBRIDIZATION USING PCR-GENERATED DNA PROBES LABELED WITH DIGOXIGENIN BY NICK TRANSLATION

Northern blot hybridization is one of the most convenient methods of d etecting an mRNA. Nonradioactive Northern blotting using digoxigenin ( DIG) is becoming widely applied because it is rapid and safe. Previous studies have indicated that DIG-labeled RNA probes are suitable for N orthern blot hybridization. Here, the application of PCR-generated dou ble-stranded DNA probes labeled with DIG by nick translation is descri bed. DNA probes were synthesized by PCR, then labeled with DIG by nick translation. Northern blot hybridization was performed using the DIG- labeled DNA probes, and the signals were detected by means of a chemil uminescent reaction. A low amount of DIG-dUTP in the labeling reaction resulted in excellent Northern blots with low background Densitometri c analysis of the blots showed that the mRNA concentrations could be d etermined by densitometric analysis. The sensitivity of the DIG-Northe rn system was comparable to Northern blotting using P-32 and was suffi ciently sensitive to defect low-abundance mRNA.