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

We have developed a nonisotopic RNase protection assay using RNA probes that are dual-labeled with biotin and fluorescein for detection. This system utilizes capture of the protected RNA probe hybrids to streptavidin-coated membranes attached to plastic dipsticks, complexing of anti-fluorescein–urease conjugate with the labeled RNA probe, and quantitative detection of the membrane-bound complex by a potentiometric silicon sensor. The dual-label RNase protection (RP) assay was capable of measuring β-actin mRNA in cellular RNA samples at the 27- to 45-amol level (10–17 pg) with high precision (%CV < 7). We have used this method to quantitate the levels of erbB-2 mRNA in the human tumor cell lines SKBR-3, SKOV-3, and MCF-7. The levels of erbB-2 mRNA in these cells were 105, 190, and 0.9 amol per microgram of cellular RNA, respectively. The dual-label RP method should be useful for measuring the mRNA expression for other erbB-2 homologs such as erbB-3 and erbB-4 in tumor cells and tissues and can be a generally useful mRNA quantitative method for laboratories wishing to minimize radioisotope use.