An improved method for precise quantitation of cellular and tissue apolipoprotein A-I mRNA levels by use of an internal standard.
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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Upjohn Company, Kalamazoo, MI 49001.
Marotti
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Melchior
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D000818:Animals; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D004277:DNA, Single-Stranded / Q000235:genetics; D008099:Liver / Q000032:analysis; D008252:Macaca fascicularis; D008722:Methods; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013997:Time Factors
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D015719:Single-Strand Specific DNA and RNA Endonucleases