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 beta-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.
Chan
S D
SD
Molecular Devices Corporation, Sunnyvale, California 94089, USA.
Dill
K
K
Blomdahl
J
J
Wada
H G
HG
D000199:Actins / Q000235:genetics; D000818:Animals; D001710:Biotin; D001943:Breast Neoplasms / Q000737:chemistry / Q000235:genetics; D016466:CHO Cells; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D006224:Cricetinae; D005260:Female; D019793:Fluorescein; D005452:Fluoresceins; D018734:Genes, erbB-2; D006801:Humans; D015336:Molecular Probe Techniques; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D012260:Ribonucleases; D014407:Tumor Cells, Cultured
D000199:Actins; D005452:Fluoresceins; D015347:RNA Probes; D012333:RNA, Messenger; D001710:Biotin; D012260:Ribonucleases; D019793:Fluorescein