Application of dual-digitonin-pulse perfusion to the study of hepatic mRNA zonation.
Heterogeneous zonation of hepatic protein expression over the liver lobule has been recognized by using several analytical techniques, including microdissection, selective cell isolation, immunohistochemistry and hybridization of mRNA in situ. We previously employed the technique of dual-digitonin-pulse perfusion for the highly selective collection and analysis of periportal and perivenous soluble protein. In the present work we have now documented the feasibility of the application of this technique to the study of zonal distribution of mRNA. By using a split-stream design, both protein and RNA fractions can be simultaneously collected from hepatic zones. High-quality RNA (average yield approximately 9-33 micrograms of total RNA per mg of eluted protein) is obtained for analysis. As analysed by immunoblotting and Northern-blot analysis, the zonal distribution of several important cytosolic metabolic enzymes and their mRNAs can be documented. This technique is also applicable to the study of mRNAs for organelle- and membrane-associated proteins that are not recoverable with this digitonin-lysis technique. The application of this experimental technique should allow further molecular insight into the mechanisms underlying zonation of hepatic function.
Witters
L A
LA
Department of Medicine, Dartmouth Medical School, Hanover, NH 03755.
Christensen
A
A
Fassihi
K
K
King
A N
AN
Widmer
J
J
Quistorff
B
B
D000818:Animals; D015870:Gene Expression; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D010477:Perfusion; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D006903:Hydroxymethylglutaryl CoA Reductases; D005941:Glucokinase; D011972:Receptor, Insulin; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D005974:Glutamate-Ammonia Ligase