A sensitive, nonradioactive method for the detection of a low level of apoptosis in rat ovary and human placenta. Technical note.
Apoptosis is often characterized by internucleosomal DNA fragmentation, which is classically visualized in standard agarose gel electrophoresis. However, this technique is not sufficiently sensitive for the detection of a modest apoptotic level in intact tissues. We developed a sensitive, nonradioactive method for the qualitative and quantitative analyses of apoptotic DNA fragmentation in intact tissues. An ultrasensitive chemiluminescent substrate, CDP-Star, was used for the visualization of digoxigenin (DIG)-labeled DNA fragments, and banding patterns were densitometrically quantified. Serially diluted DNA samples from rat ovaries were labeled with DIG. As a result, only 1.56 ng of DNA could be analyzed for the presence of apoptotic DNA cleavage. The sum intensity of these bands increased almost linearly by increasing the amount of labeled DNA. In human placental tissues, clear apoptotic DNA ladders were visualized by this method, and the quantification of apoptotic DNA fragmentation was also possible. This new method provides a nonradioactive, highly sensitive and semiquantitative analysis of apoptotic DNA fragmentation and may be highly useful for the study of a low, physiological level of apoptosis.
Furuta
I
I
Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, Sapporo, Japan. furuchan@med.hokudai.ac.jp
Yamada
H
H
Sakuragi
N
N
Fujimoto
S
S
D000818:Animals; D017209:Apoptosis; D001345:Autoradiography; D004247:DNA / Q000032:analysis; D053938:DNA Fragmentation; D004076:Digoxigenin; D005260:Female; D006801:Humans; D020287:In Situ Nick-End Labeling; D008163:Luminescent Measurements; D010053:Ovary / Q000737:chemistry* / Q000166:cytology; D010920:Placenta / Q000737:chemistry* / Q000166:cytology; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley
D004247:DNA; D004076:Digoxigenin