Quantitation of mRNA. An RT-PCR method was used to quantify mRNA levels in cells treated with antisense oligonucleotide (Oligo AS) or AS mismatch control (ASM). Cells were transfected with oligos in the presence of Lipofectin (7 m g/ml; Life Technologies, Gaithersburg, MD) and 1% FBS for various times prior to analysis of mRNA levels. Total RNA was extracted by using the Trizol reagent from Invitrogen, quantified by UV spectrophotometry, and used to create cDNA by using the SuperScript RT-PCR kit from Invitrogen. For each reverse transcription (RT) reaction, 5 m g of RNA was mixed with 1 m l of dNTP (10 mM) and 1 m l of Oligo(dT)_12-18 (0.5 m g/m l) and incubated for 5 min at 65°C. The following were added to the reaction mixture: 2 m l of 10´ RT buffer (200 mM Tris·HCl, pH 8.4/500 mM KCl), 2 m l 0.1 M DTT, and 1 m l of RNase inhibitor RNaseOut (40 units/m l). The mixture was then incubated at 42°C for 2 min followed by addition of 1 m l of RT enzyme (200 units/m l) to each tube. The reaction was allowed to proceed at 42°C for 50 min, followed by reaction termination at 70°C for 15 min. cDNA was stored at –80°C before use.

PCR of the cDNA samples from the RT reaction was accomplished in a volume of 50 m l. The primers for each gene are listed in Table 1. The reaction mixtures for coamplification of mouse double minute 2 (MDM2), P53, BAX, and E2F transcription factor 1 (E2F1) with b -actin contained 1´ PCR buffer (50 mM KCl/10 mM Tris·HCl, pH 9.0/0.1% Triton X-100/2.5 mM MgCl₂), 0.2 mM dNTP, primers (0.4 m M for MDM2 and P53, 0.2 m M for BAX, and 0.4 m M for E2F1), b -actin primers (0.04 m M for MDM2, BAX, and E2F1 coamplification and 0.08 m M for P53 coamplification), 2 units of Taq DNA polymerase (Promega) and 4 m l of cDNA from RT reaction (diluted 1:4). To coamplify WAF1, RB, and BCL2, the reaction mixtures were 1´ PCR buffer (50 mM KCl/10 mM Tris·HCl, pH 9.0/0.1% Triton X-100/1.5mM MgCl₂), primers (0.36 m M WAF1 and BCL2 and 0.2 m M for RB), b -actin primers (0.04 m M), 2 units of Taq DNA polymerase, and 2 m l of cDNA from RT reaction (diluted 1:4). Prior to PCR cycles, all samples were incubated at 94°C for 5 min. The cycle sequences for each target gene were programmed as follows: MDM2 (32 cycles): 94°C (1 min), 62°C (1 min), 72°C (2 min); P53 (31 cycles): 94°C (1 min), 48.7°C (30 sec), 72°C (1 min 30 sec); WAF1 (24 cycles): 94°C (45 sec), 62°C (1 min), 72°C (1 min 20 sec); RB/BCL2 (33 cycles): 94°C (45 sec), 57.3°C (1 min), 72°C (1 min); BAX (31 cycles): 94°C (1 min), 59.9°C (1 min), 72°C (1 min); E2F1 (39 cycles): 94°C (1 min), 54.5°C (1 min 15 sec), 72°C (1 min 10 sec) . Fifteen microliters of the amplified products was run on a 0.8 % agarose gel and bands were visualized with ethidium bromide. The density of each band was analyzed by a densitometry measurement (Kodak Imaging Analysis 1D3.6 Program, Eastman Kodak).

Quantitative Analysis of the Mode of Action After Combination Treatment of Oligo AS or ASM with Paclitaxel. The relative ratio ([2]/[1] or [3]/[1]) can be used to illustrate the potential additive or synergistic effects when the mixed-backbone oligos (MBOs) were given in combination with paclitaxel. When the ratio for combination therapy (AS or ASM + paclitaxel) was <100% (compared with paclitaxel alone), an effect of the MBO was indicated. If the ratio for combination therapy was the same as that of MBO alone, an additive effect was indicated. If the ratio for combination therapy was significantly smaller than that of MBO treatment alone, a synergistic effect was indicated. Based on this model, Oligo AS had additive effects on paclitaxel treatment.