5156 Nucleic Acids Research, 2007, Vol. 35, No. 15 microRNAs, the luciferase units were compared to those obtained when the target was co-transfected with irrele- vant shRNA and the microRNA controls. Similarly the impairment of Luc activity by the synthetic oligonucleo- tides was determined by comparing the luciferase units with those obtained when the luciferase target was transfected with only the irrelevant synthetic siRNA. It is important to normalize the experimental samples to their speciﬁc controls since si, sh or microRNA constructs co-transfected with a target could non-speciﬁcally alter the results by increasing or decreasing the expression of that target. All the experiments were normalized for transfec- tion eﬃciency, carried out in duplicate, and repeated a minimum of three times. Diﬀerent amounts of Bluescript plasmid (Stratagene) were added to all the transfections to obtain the same total amount of DNA transfected in each experiment, which corresponded to 400 ng per reaction (1 ml Lipofectamine-2000) except for the transfections in the HCT116-GFPmiR21 cell line in which the total amount of transfected DNA was 800 ng (2 ml Lipofectamine-2000; no Bluescript was added). The cells were analyzed for luciferase or EGFP expression 48 h following transfections. Luciferase assays A total of 293 cells were seeded in 24-well plates and transfected the next day at 80% conﬂuency. Twenty-four or 48 h post-transfection, the medium was removed and cells were washed once with DPBS (Dulbecco’s Phosphate Buﬀer Saline, Cellgro). One hundred microliter of 1 Passive Lysis Buﬀer (Promega) was then added to each well. Cells were lysed at room temperature with gentle shaking for 20 min. Ten microliter of cell lysates were assayed for dual luciferase activity according to the manufacturer’s instructions (Dual Luciferase Reporter System, Promega). Changes in the levels of the FLT were calculated after normalization against the Renilla lucifer- ase units. When the Renilla luciferase was used as the target, changes in expression were calculated relative to ﬁreﬂy luciferase (transfection control). The luminescence was determined using a Veritas luminometer (Turner BioSystems, Inc. Sunnyvale, CA, USA). Results are presented in relative light units and the SD is indicated with error bars as shown in the graph data. Northern analyses Total RNA was isolated using RNA STAT-60 (TEL-TEST B Inc., Friendswood, TX, USA) according to the manufacturer’s instructions. Fifteen microgram of total RNA were fractionated in 8 M–6% PAGE, and transferred onto a Hybond-N+ membrane (Amersham Pharmacia Biotech). 32P-radiolabeled 21-mer probes complementary to the siRNA-antisense sequences were used for the hybridization reactions, which were performed for 16 h at 378C. A 21-mer DNA oligonucleotide was electrophoresed alongside the RNA samples and used as a size marker and hybridization control (not shown). For TRBP detection, 30 mg RNA was fractionated on a 1% MOPS agarose gel, transferred on a Hybond-N+ membrane and hybridized with a 32P labeled probe complementary to TRBP overnight at 378C. Immunodepletion assay Conﬂuent HEK293 cells in a 10 cm dish were harvested with 1 PBS after trypsinizing and resuspended in 0.5 ml of Buﬀer D (20 mM HEPES, pH 7.9, 0.2 mM EDTA, 0.5 mM DTT, 50 mM KCl, 10% Glycerol and 0.2 mM PMSF). The cells were sonicated for 15 s in ice water, and the supernatant was collected following 5 min centrifugation. The immunodepletion was carried out by incubating 100 ml of the extract with 30 ml of anti-TRBP antibody (27) or 30 ml (2 mg/ml) of anti-b-tubulin (Abcam Inc., Cambridge, MA, USA) antibody. Following overnight incubation at 48C, 30 ml of protein A agarose beads (Upstate USA, Inc. Chicago, IL, USA) were added and incubated for 1.5 h at 48C with gentle agitation. Following the removal of the beads by a brief centrifuga- tion, 3 ml of the supernatant were resolved in a 10% SDS-PAGE for western blot analyses, and 10 ml were used for the electrophoretic mobility gel shift assay. The pellet beads were boiled with 25 ml of 2 SDS loading buﬀer and 10 ml were resolved alongside the supernatant sample for western blot analyses. After transferring proteins overnight, the PVDF membranes were blocked with 5% milk in 1 PBS for 2 h at room temperature and washed with 1 PBST for 10 min three times. Primary and secondary antibody incubations were carried out each for 1.5 h in 5% milk in 1 PBST at room temperature followed by washing with 1 PBST for 10 min three times. The membranes were rinsed with 1 PBS for 5 min before drying. The secondary antibody labeled with a 700 channel dye, anti-rabbit-680 nm, was used to detect rabbit polyclonal anti-TRBP Ab672 and b-tubulin. Gel shift assays For each reaction, 10 ml of the TRBP or b-tubulin immunodepleted cell extract was incubated with 10 fmol of 50 end-labeled siRNA for 30 min at room temperature. The samples were mixed with 2 native gel loading dye and resolved in a 5% non-denaturing polyacrylamide gel for 3 h at 200 V at 48C. The HEK 293 total cell extract and the tubulin immunodepleted cell extract were used as controls. P24 assays The human T cell line CEM was maintained in RPMI 1640 medium supplemented with 10% FBS. A total of 1 106 transduced CEM T cells were infected with HIV-1 strain IIIB at an MOI of 0.01. After overnight incubation, cells were washed three times with Hanks’ balanced salts solution and cultured in medium with R10 (RPMI 1640 plus 10% FBS). At designated time points, culture supernatants were collected for p24 analyses (HIV-1 p24 antigen EIA kit, Beckman Coulter Inc.).