Internal cRNA standards for quantitative northern analysis.
We report a simple method using copy RNA (cRNA) internal standards for quantitative Northern hybridization. This was accomplished by synthesis of a full-length or "half"-length cRNA and mixing these RNA internal standards with samples to be tested for the abundance of a given mRNA. Both full-length and truncated cRNAs are detected in Northern analysis by the nucleic acid detection probe (which can be labeled with 32P or biotin), and the known amount of the truncated cRNA is compared to the homologous mRNA present in the specimen being examined. We demonstrate the usefulness of this method by measuring the expression of renin in rat kidney with ureteral obstruction and angiotensin II receptor (AT1-R) mRNA in kidneys from spontaneously hypertensive rats. It was found that this method of preparing cRNA internal standards successfully controlled for variables (such as differences in loading, transfer and hybridization efficiency) that often frustrate efforts to use Northern analysis as a quantitative tool and enabled direct estimation of absolute mRNA amount present in samples. This technique may have wide applicability and permit a more quantitative use of Northern analysis.
Wang
S
S
Department of Medicine, Emory University, Atlanta, GA 30322.
Murtagh
J J
JJ
Luo
C
C
Martínez-Maldonado
M
M
D000818:Animals; D015152:Blotting, Northern / Q000379:methods*; D012313:RNA / Q000032:analysis*; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D011945:Receptors, Angiotensin / Q000235:genetics; D012015:Reference Standards; D012083:Renin / Q000235:genetics
D018075:RNA, Complementary; D012333:RNA, Messenger; D011945:Receptors, Angiotensin; D012313:RNA; D012083:Renin