nucleotides on the function of agRNAs. After examining over 120 duplexes, we identify several chemically modiﬁed compounds that retain partial or full activity. MATERIALS AND METHODS Double-stranded RNAs RNAs were synthesized by Alnylam Pharmaceuticals or SIGMA Custom Products using standard protocols, or were purchased from Integrated DNA Technologies. The concentrations of single strands were determined by measuring their absorbance at 260 nm (A260). Complementary single strands were combined in equimolar ratios and diluted to 20 mM stock solutions in 2.5 phosphate-buﬀered saline. These duplexes were annealed by heating to 95 C and slowly cooled to room temperature in a heating block or thermal cycler. Duplex integrity was tested by a UV thermal melt experiment as described below. Duplexes were stored at 20 C. Cell culture T47D and MCF7 cells (American Type Culture Collection) were maintained in RPMI-1640 media (Sigma) supplemented with 10% (v/v) FBS, 0.5 MEM non-essential amino acids, 0.4 units ml 1 bovine insulin, 10 mM HEPES and 1 mM sodium pyruvate. Cells were cultured at 37 C and 5% (v/v) CO2. Lipid-mediated transfection Cells were plated at 110 000–160 000 cells per well in six-well plates (Costar) in RPMI. A higher number of cells was typically seeded for RNA analysis than for protein analysis. Transfections were performed after 1–2 days, when the cells had adhered and attained about 20–30% conﬂuence. Lipofectamine RNAiMAX cationic lipid (1.1 ml/well, Invitrogen) and RNA (31 pmol/well) were individually diluted in Opti-MEM (Invitrogen), then combined and allowed to incubate for 20 min (volume 250 ml/well) before pipetting into cell-culture wells containing 1 ml fresh Opti-MEM (ﬁnal volume 1.25 ml, 25 nM duplex RNA for all transfections except dose response experiments). The transfection solution was replaced with RPMI after 24 h, and media were changed again 2 days later. For dose response experi- ments, the ratio of lipid to RNA was kept constant, and the concentration of RNA was varied from 0 to 50 nM. For competition experiments, the cells were passaged into new plates on Days 2 or 3 after transfection. The second transfection was carried out on Day 4 after the ﬁrst transfection under identical conditions, and protein was harvested on Days 7 or 8. The negative control duplex Neg1 used in these experiments consisted of oligonucleo- tides 50-UCAAGAAGCCAAGGAUAAUdTdT and 50-A UUAUCCUUGGCUUCUUGAdTdT. RNA harvest and quantitative real-time PCR Total RNA from treated MCF7 or T47D cells was har- vested 3 days after transfection. After washing with PBS, TRI reagent (1 ml, Sigma) was added to each well, Nucleic Acids Research, 2010, Vol. 38, No. 15 5243 incubated for several minutes, mixed well and transferred to a 1.5-ml microcentrifuge tube. Chloroform (250 ml) was added and the mixture was shaken vigorously for 1 min, then centrifuged at 12 500 rpm for 15 min. The clear aqueous layer was transferred to a new 1.5-ml microcentrifuge tube, avoiding any of the interphase or organic layer. Addition of cold isopropanol (500 ml) followed by shaking, incubation at 20 C (at least 10 min), and centrifugation (12 500 rpm, 15 min, 4 C) gave a pellet, which was washed with 75% ethanol, dissolved in nuclease-free water (20 ml), and quantitated by the A260 of an aliquot (2 ml in 200 ml TE buﬀer). Samples of 2 mg RNA were removed and treated with DNase I (Worthington). After heat-inactivation of the DNase enzyme (75 C, 10 min), the samples were reverse transcribed using random hexamer primers with the High Capacity cDNA Archive kit (Applied Biosystems). Quantitative real-time PCR (qPCR) was used to measure RNA levels with 30–100 ng of cDNA (carried out in triplicate, and repeated for at least two independent transfections). For analysis of PR mRNA, we used a primer-probe assay (IDT) consisting of primers 50-CTTA CCTGTGGGAGCTGTA-30 and 50-GCACTTTCTAAG GCGACATG-30 as well as probe 50-CTGCCCTTCCAT TGCCCTCTT-30. For analysis of promoter-derived anti- sense transcripts, we used SYBRgreen qPCR with primers 50-GGAGGAGGCGTTGTTAGAAA-30 and 50-GAAGG GTCGGACTTCTGCT-30. Data were normalized to levels of GAPDH mRNA as determined by a TaqMan Gene Expression Assay (Applied Biosystems). Analysis of protein expression Protein was generally harvested from treated MCF7 or T47D cells 5 days after transfection. The cells were washed with phosphate-buﬀered saline (PBS), trypsinized, transferred to 1.5-ml microcentrifuge tubes and centrifuged (3500 rpm, 15 min). The pellets were washed with cold PBS and gently lysed by addition of 50 ml lysis buﬀer [50 mM Tris–HCl (pH 8.0), 120 mM NaCl, 0.5% Nonidet P40, 1 mM EDTA, 1 mM DTT]. Protease inhibitor cocktail set 1 (Calbiochem) was added to the lysis buﬀer just before use. After a freeze-thaw cycle to ensure complete lysis, the lysates were centrifuged to pellet insoluble cell debris and cell lysate solutions were transferred to new microtubes. Protein concentrations were calculated using the BCA assay (Thermo Scientiﬁc). Western blots were performed on cell lysates (30 mg protein per lane). Proteins were separated by SDS–PAGE using 7.5% Tris–HCl acrylamide gels, then transferred onto nitrocellulose membranes. After blocking the membrane (5% milk in PBS-T) for 1 h, the upper portion of the membrane was treated with PR monoclonal primary antibody (Cell Signaling, 1:1000 dilution in PBS-T, 15 h) and the lower portion was treated with anti–b-actin (Sigma, 1:10 000 dilution, 1 h) as an internal control. Protein was visualized using HRP-conjugated sec- ondary anti-mouse (Jackson Immunolabs, 1:5000 for PR and 1:20 000 for actin, 30–45 min) The membranes were then brieﬂy treated with West Pico SuperSignal develop- ing solution (Thermo Scientiﬁc) before exposing them to