A rapid colorimetric in situ mRNA hybridization technique using hyperbiotinylated oligonucleotide probes for analysis of mdr1 in mouse colon carcinoma cells.
We describe the development of a rapid colorimetric in situ hybridization technique utilizing oligonucleotide probes labeled with six biotin molecules at the 3' end to detect mdr1 in mouse colon cancer cells growing in culture and in vivo. mRNA integrity was verified by the use of a multibiotinylated poly d(T) oligonucleotide, and the specificity of the reaction was confirmed by use of labeled sense and anti-sense probes in serial cryostat sections and cultured cells. The multiple biotin label produced a strong signal after a short hybridization time. Avidin-alkaline phosphatase detection and the capillary technology used in the Microprobe Accelerated System allowed completion of the procedure in less than 5 hr. Excellent correlations with the MDR phenotype of the cells, Northern blot analysis, and immunohistochemistry recommend this procedure for identifying cells that express the MDR phenotype in culture and in vivo.
Bucana
C D
CD
Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Radinsky
R
R
Dong
Z
Z
Sanchez
R
R
Brigati
D J
DJ
Fidler
I J
IJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D016375:Antisense Elements (Genetics); D001483:Base Sequence; D015152:Blotting, Northern; D003110:Colonic Neoplasms / Q000235:genetics*; D004351:Drug Resistance; D005629:Frozen Sections; D017403:In Situ Hybridization; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics; D015345:Oligonucleotide Probes; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016375:Antisense Elements (Genetics); D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D012334:RNA, Neoplasm