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1Department of Anesthesiology and Critical Care Medicine, 2Center for Clinical Pharmacology, and 3Center for Environmental and Occupational Health and Toxicology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 The quantitation of mRNA expression has the potential to be of critical value in a clinical setting but, to date, has been limited by current quantitative PCR techniques. Many techniques evaluate the relative differences in target amounts rather than determine the copy number, and there are safety and time concerns associated with 32p labeling of PCR products. The recently developed fluorescein-antifluorescein-based enzyme-linked oligonucleotide sorbent assay (FAF-ELOSA) technique overcomes many of these difficulties. We describe a comparison of results obtained by the 32p-labeled PCR/Southern blotting technique to the FAF-ELOSA. Key features of the FAF-ELOSA technique include quantitation by nonradioactive detection based on relative amounts of PCR product and a synthesized standard target. It is reproducible from a single cDNA preparation or from samples derived from separate preparations. The technique can easily be applied to clinical samples for the rapid determination of many different specific mRNA expression studies. We are currently applying this technique for the detection of CYP450 mRNA expression in a variety of human tissues from control and cancer patient populations. Since the PCR method was first introduced in 1985, (1~ the combination of reverse transcription (RT) with PCR (RTPCR) has increasingly been utilized to study gene expression. (2-s~ However, the use of mRNA quantitation as a clinical tool has been hampered by several limitations of currently utilized techniques, including the 32P-labeled PCR/Southern blotting technique. First, the 32P-labeled technique is considered semi-quantitative rather than quantitative because of the narrow linear range of film exposure and scanning densitometry. Also, the use of 32p precludes automated mass analysis of clinical samples. Third, the quantitation of the target mRNA is dependent on the accuracy of the initial RNA quantitation and the relative efficiency of the RT. Finally, the blotting technique requires time periods that are not compatible with same day, diagnosis-directed therapeutic interventions. In addition to varying cycle number, using amplification of a control gene in parallel, or using a competitive template as an internal control, (6) several investigators have developed biotin, fluorescein, and streptavidin techniques to address these shortcomings. Syvanen et al. (7) hybridized biotinylated PCR products to an oligonucleotide probe and immobilized the complex to avidin-coated beads. Saiki et al. (1~ showed that a PCR product could be identified specifically by hybridization to an oligonucleotide probe immobilized on a nylon membrane. The same principle has been applied to microtiter plate technology using ELOSA by Inouye and Hondo (a~ and Keller et al. (9'1~ Recently it was demonstrated that a double-stranded PCR product obtained with a biotinylated primer and a fluorescein-labeled primer could be detected upon immobilization in an avidin-coated plate and conjugation with a FAF-ELOSA. ( ~ This technique has been successfully used to measure drug resistance of human immunodeficiency virus type 1 (HIV-1) isolates by determining relative HIV-1 copy number in the supernatant of cultured peripheral blood monocytes. (12~ To our knowledge, none of these studies have used the FAF-ELOSA protocol to quantitate mRNA, nor have they compared this quantitation with standard 3zp methodology. There have been reports that 32p incorporation into the PCR product shows a good correlation with slot blot (3~ and Northern blot (4~ for the quantitation of the multidrug resistance gene mdr-1 expression and with in situ hybridization for progesterone receptor mRNA. ~ In the present work we compare the relative sensitivity of the FAF-ELOSA with the sensitivity of the 32p-labeled PCR/Southern blotting technique in quantitation of PCR products used to measure rat and human actin mRNA. Cytochrome P450 CYP2D6 and CYP2E1 mRNA expression in human hepatic tissue are evaluated as an example of applying this technique to the analysis of specific mRNAs of interest in clinical samples.

genome research

Comparison of the enzyme-linked oligonucleotide sorbent assay to the 32P-labeled PCR/Southern blotting technique in quantitative analysis of human and rat mRNA.