5244 Nucleic Acids Research, 2010, Vol. 38, No. 15 ﬁlm. All analogs were tested in at least two independent transfections, of which one representative western blot is shown. Determination of melting temperature (Tm) Annealed siRNA duplexes were diluted to 0.8 mM in 0.1 M phosphate buﬀer, pH 7.4 (ﬁnal volume 500 ml) and transferred to quartz cuvettes. The solutions were topped with mineral oil (200 ml) and heated from 18 to 99 C at 1 C/min while monitoring changes in A260 using a Cary 100 Bio spectrophotometer. Samples were then re-annealed in the spectrophotometer and the experiment was repeated several times. Tm values were calculated as the ﬁrst derivatives of the plot of A260 vs temperature, and the results from three experiments were averaged. Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) was performed essentially as described (29). Two or three 150-mm dishes of T47D or MCF7 cells were reverse transfected with 25 nM of native agRNA, modiﬁed agRNA or luciferase control duplex siGL2 (30). Media were changed on Day 2 and the cells harvested on Day 3. To crosslink and harvest nuclei, the cells were treated with 1% formaldehyde in PBS (10 ml) for 10 min, then the crosslinking reaction was quenched with glycine (0.125 M ﬁnal concentration, at least 5 min). Cells were collected and nuclei were isolated by treatment with cold hypotonic lysis buﬀer (4 ml) containing 10 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2 and 0.5% Nonidet P40. The pelleted nuclei were lysed with a buﬀer containing 1% SDS, 10 mM EDTA, 50 mM Tris–HCl (pH 8.1) and 1 complete protease inhibitor cocktail (Roche). DNA was sonicated on ice to an average fragment size of 500 bp. The lysate was then centrifuged to remove insoluble cell debris and the supernatant precleared with Protein G Plus/Protein A Agarose suspension (Calbiochem). An aliquot of the cleared lysate (100 ml) was diluted to 1 ml in buﬀer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.1, 167 mM NaCl and 1 complete protease inhibitor cocktail (Roche)]. Mouse anti-RNAPII IgG (Millipore, Catalogue #05-623, 3–6 mg) or normal mouse IgG (Millipore Catalogue #12-371, 3–6 mg) was added and the solution rotated at 4 C overnight. Protein G Plus / Protein A agarose suspen- sion (30–60 ml) was added to precipitate the antibody complexes and the beads were washed as described (29). Complexes were eluted using 2 250 ml of NaHCO3 (0.1 M) containing 1% SDS. NaCl was added (ﬁnal con- centration 200 mM) and the samples heated to 65 C for at least 2 h to reverse crosslinks. The samples were then treated with RNase A (1 ml) at 37 C for 30 min, followed by Tris–HCl (pH 7.0, 20 ml), EDTA (0.5 M, 10 ml) and proteinase K (20 mg) at 42 C for 45 min. DNA was isolated by phenol-chloroform extraction and ethanol pre- cipitation. Pellets were diluted to 50 ml in water and 5 ml was used per qPCR reaction. qPCR primers used at the PR promoter were Fwd 50-CCTAGAGGAGGAGGCGT TGT and Rev 50-ATTGAGAATGCCACCCACA. Levels of RNAPII at the GAPDH promoter were also measured with primers Fwd 50-TACTAGCGGTTTTACGGGCG and Rev 50-TCGAACAGGAGGAGCAGAGAGCGA and were used for normalization. Each sample was normalized to a corresponding 2% input sample. Statistics, dose response proﬁles, and curve ﬁtting Error bars shown on qPCR graphs correspond to standard deviations from triplicate measurements of at least two independent transfections; they include the error from the qPCR measurement as well as from diﬀer- ences between the transfections. For Figures 2–7 we also examined whether RNA expression levels were signiﬁcant- ly diﬀerent from the mismatch control by conducting a two-tailed unpaired Student’s t-test with equal variances, and for this test we considered only biological replicates (using averages of the triplicate qPCR measurements for each). Signiﬁcance levels of P-values are: *P < 0.05, **P < 0.01 and ***P < 0.005. Dose responses were ﬁt to the model equation y = 100[1 – xm/(nm+xm)], where y is the percent of target (protein or mRNA) remaining, x is the concentration of agRNA in nanomolars. The regression was ﬁt by optimizing m and n using SigmaPlot 11, and n was taken as the IC50 value. RESULTS AND DISCUSSION Experimental design We focused on the PR gene because it is a well-characterized target for agRNAs (20,23,25,28). In T47D breast cancer cells, which express a high level of PR, PR expression can be dramatically reduced by agRNA PR-9 that overlaps the start site from –9 to +10 (20,21). In MCF7 breast cancer cells, which express PR at a lower basal level, its expression can be increased by agRNA PR-11 targeting the region from –11 to +8 (23). PR-11 can also activate gene expression in T47D cells but activation becomes most apparent when the cells are grown under conditions that repress basal expression of PR (23). Whether activation or repression is observed depends primarily on the basal expression level of PR and the target sequence of the agRNA. While PR-9 and PR-11 are benchmark agRNAs used for most experiments, we also examined other duplexes. For some modiﬁcations we tested analogs of silencing agRNA PR-26 (–26 to –8) or activating agRNA PR-22 (–22 to –4) to allow more general insights into gene modu- lation. To permit comparisons with an RNA that engages the post-transcriptional silencing pathway, we used modiﬁed analogs of PR2526, a siRNA that is complemen- tary to PR mRNA from +2526 to +2544. As negative control we used MM4, a duplex containing four mismatches with respect to PR-9. Duplex and single- stranded oligomers were introduced into cells by standard transfection protocols using cationic lipid. Expression was analyzed by western analysis of PR protein and qPCR of PR mRNA. We use descriptive compound identiﬁers (Table 1). Each type of modiﬁed duplex is assigned two uppercase letters.