Direct solution hybridization of guanidine thiocyanate-solubilized cells for quantitation of mRNAs in hepatocytes.
The sensitivity of direct solution hybridization of hepatocytes solubilized in guanidium thiocyanate (GuSCN) for detecting alpha 1-acid glycoprotein and albumin mRNAs was studied. The sensitivity of detection was inversely correlated with the DNA concentration. Raising the hybridization temperature from 20 to 37 or 50 degrees C (with formamide) increased the hybridization efficiency three- to fourfold in cell lysates with a high DNA concentration (1 microgram/microliter), whereas the hybridization efficiency was already maximal at 20 degrees C in diluted samples. It was most important to normalize all hybridization reactions with an internal standard, such as sense mRNA, because of the great variation in hybridization efficiency from one cell preparation to another depending on the DNA concentration. Direct hybridization of GuSCN cell lysates labeled in vivo with [6-14C]orotic acid was more efficient than hybridizing equivalent amounts of purified [6-14C]-labeled RNA, perhaps because of greater mRNA integrity and/or better recoveries of mRNA in GuSCN cell lysates. Therefore, direct solution hybridization of GuSCN-solubilized hepatocytes, which avoids the problem of RNA purification, appears to be a rapid, sensitive, and reliable method for quantifying mRNA in hepatocytes.
Kaabache
T
T
Laboratoire de Biologie Cellulaire, INSERM U 327, Faculté de Médecine Xavier Bichat, Université Paris, France.
Barraud
B
B
Feldmann
G
G
Bernuau
D
D
Lardeux
B
B
D000418:Albumins / Q000235:genetics*; D000818:Animals; D002250:Carbon Radioisotopes / Q000032:analysis; D002462:Cell Membrane / Q000187:drug effects; D004247:DNA / Q000032:analysis; D003902:Detergents / Q000494:pharmacology*; D006146:Guanidines / Q000494:pharmacology*; D007553:Isotope Labeling / Q000379:methods; D008099:Liver / Q000737:chemistry* / Q000166:cytology; D008297:Male; D009693:Nucleic Acid Hybridization; D009961:Orosomucoid / Q000235:genetics*; D009963:Orotic Acid; D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012996:Solutions; D013696:Temperature; D013861:Thiocyanates / Q000494:pharmacology*
D000418:Albumins; D002250:Carbon Radioisotopes; D003902:Detergents; D006146:Guanidines; D009961:Orosomucoid; D015347:RNA Probes; D016372:RNA, Antisense; D012333:RNA, Messenger; D012996:Solutions; D013861:Thiocyanates; C054436:guanidine thiocyanate; D009963:Orotic Acid; D004247:DNA