Nonisotopic Quantitation of mRNA Using a Novel RNase Protection Assay: Measurement of erbB-2 mRNA in Tumor Cell Lines (original) (raw)

1996, Analytical Biochemistry

partially degraded RNA. The assay involves hybridiza-We have developed a nonisotopic RNase protection tion of RNA samples to radiolabeled antisense RNA assay using RNA probes that are dual-labeled with bioprobe in solution, followed by digestion of unhybridized tin and fluorescein for detection. This system utilizes single-stranded RNA with RNases. The protected racapture of the protected RNA probe hybrids to strepdioactive hybrid fragments are recovered by ethanol tavidin-coated membranes attached to plastic dipprecipitation and analyzed by denaturing polyacrylsticks, complexing of anti-fluorescein-urease conjuamide gel electrophoresis and autoradiography (1). Algate with the labeled RNA probe, and quantitative ternatively, the hybridized nucleic acids are captured detection of the membrane-bound complex by a potenonto nitrocellulose or nylon membrane, and radioactivtiometric silicon sensor. The dual-label RNase protecity is then detected by liquid scintillation counting (2). tion (RP) assay was capable of measuring b-actin One major drawback of this technique, however, is the mRNA in cellular RNA samples at the 27-to 45-amol requirement for radioactivity. Because of the increaslevel (10-17 pg) with high precision (%CV õ 7). We ing concerns about the health hazards and high dishave used this method to quantitate the levels of erbBposal cost of radioactive waste, we have developed a 2 mRNA in the human tumor cell lines SKBR-3, SKOVnonisotopic RP assay using RNA probes dual-labeled 3, and MCF-7. The levels of erbB-2 mRNA in these cells with biotin and fluorescein, with subsequent detection were 105, 190, and 0.9 amol per microgram of cellular of the protected probes using the immunoligand assay RNA, respectively. The dual-label RP method should (ILA) system (3) on a light-addressable potentiometric be useful for measuring the mRNA expression for sensor (LAPS) that has been used extensively for lowother erbB-2 homologs such as erbB-3 and erbB-4 in level detection of various analytes including total DNA tumor cells and tissues and can be a generally useful and sequence-specific DNA (4, 5). We have used this mRNA quantitative method for laboratories wishing to minimize radioisotope use.