Journal of Lipid Research, Vol 31, 727-733, Copyright © 1990 by Lipid Research, Inc.
An improved method for precise quantitation of cellular and tissue apolipoprotein A-I mRNA levels by use of an internal standard
ME Pape, KR Marotti and GW Melchior
Upjohn Company, Kalamazoo, MI 49001.
We have developed a method for the quantitation of apolipoprotein A-I
(apoA-I) mRNA by using a variation of traditional S1 nuclease analysis.
This method uses an internal standard RNA that allows a level of precision
not obtainable with traditional S1 nuclease analysis. The internal standard
RNA is synthesized in vitro using the T7 promoter from the transcription
vector, pApoAI, which contains the full length apoA-I cDNA. This RNA
molecule is identical to authentic apoA-I mRNA except for the addition of
48 bases at the 5'-end which are derived from the vector. A labeled ssDNA
probe is produced from pApoAI in such a way that solution hybridization of
the probe to a mixture of total RNA and internal standard RNA followed by
S1 nuclease digestion results in the protection of DNA fragments from
authentic and internal standard RNA, which differ in size by 48 bases. The
DNA fragments can be resolved by gel electrophoresis and quantitated. The
addition of internal standard RNA to each hybridization reaction allows for
correction of variations in hybridization and other sources of experimental
error. Using this method we demonstrate a sevenfold increase in precision
(the 90% confidence interval was reduced from +/- 186% to +/- 26% of the
mean value) when compared to traditional S1 nuclease analysis of apoA-I
mRNA in liver biopsies and hepatocytes in culture. The internal standard/S1
nuclease method can be adapted to the analysis of any mRNA.
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