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 detecting 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 Northern blot hybridization. Here, the application of PCR-generated double-stranded DNA probes labeled with DIG by nick translation is described. 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 chemiluminescent reaction. A low amount of DIG-dUTP in the labeling reaction resulted in excellent Northern blots with low background. Densitometric analysis of the blots showed that the mRNA concentrations could be determined by densitometric analysis. The sensitivity of the DIG-Northern system was comparable to Northern blotting using 32P and was sufficiently sensitive to detect low-abundance mRNA.
Sato
M
M
First Department of Internal Medicine, Kagawa Medical School, Miki-Cho, Japan.
Murao
K
K
Mizobuchi
M
M
Takahara
J
J
D000818:Animals; D015152:Blotting, Northern / Q000379:methods*; D004247:DNA / Q000097:blood; D015342:DNA Probes / Q000138:chemical synthesis*; D004076:Digoxigenin; D006801:Humans; D007031:Hypothalamus / Q000737:chemistry; D008163:Luminescent Measurements; D008214:Lymphocytes / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction; D011333:Pro-Opiomelanocortin / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013004:Somatostatin / Q000235:genetics
D015342:DNA Probes; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D013004:Somatostatin; D011333:Pro-Opiomelanocortin; D004247:DNA; D004076:Digoxigenin