Figure 1.

(A) RNA fingerprinting of MCF-7 cells. Total cellular RNA was treated with DNase, reverse-transcribed into cDNA, and amplified by PCR using upstream and downstream primers and radiolabeled [³³P]dATP. The figure depicts a portion of the autoradiograph of a 5% polyacrylamide gel electrophoresis of the PCR mixture by using the primer pair P6 and T9. Lanes 1, 3, and 5 are reaction mixtures in which cDNA diluted 1:10 was added; lanes 2, 4, and 6 represent reaction mixtures in which cDNA diluted 1:40 was added. Lanes 7 and 8 are H₂O controls, in which sterile water was added to the PCR mixture in place of cDNA. Lanes 9, 10, and 11 are RNA controls, in which 0.02 μg of cellular RNA from MCF-7/W, MCF-7/AdrVp, or MCF-7/AdrVpPR cells was added instead of cDNA. These RNA controls served to indicate contamination of the RNA with genomic DNA. The arrow indicates a PCR product representing an mRNA species overexpressed in MCF-7/AdrVp cells, compared with MCF-7/W or MCF-7/AdrVpPR cells. This product was excised from the gel for further amplification by PCR. (B) Northern blot hybridization (Upper) of mRNA from MCF-7/W, MCF-7/AdrVp, or MCF-7/AdrVpPR cells by using the 795-bp PCR product obtained from RNA fingerprinting studies and isolated by TA cloning as a probe after labeling with [³²P]dCTP (Prime-a-Gene labeling kit, Promega). To control for equivalence in sample loading, the blot was stripped and rehybridized with a radiolabeled probe for 18S RNA (Lower).