ASON-DNAzyme Complex Figure 6. Semi-quantitative RT-PCR and Western blot analysis of the mechanism of ASONBulge-DNAzyme. A. RT-PCR of eGFP. B. RT- PCR of b-actin. C. Western blot of eGFP. doi:10.1371/journal.pone.0018629.g006 cDNA was synthesized using 4 ml of the total RNA, 4 ml of 56reverse transcriptase buffer, 1 ml of 10 mM dNTPs, 1 ml of eGFP-R1 primer (10 mM), 1 ml of b-actin-R1 primer (10 mM), 1 ml of RNase inhibitor (20 U/ml), 1 ml of MMLV reverse transcriptase (5 U/ml) and 7 ml of RNase-free H2O in a total volume of 20 ml for 1 h at 42uC. The PCR conditions were as follows: 1 cycle at 94uC for 5 min; followed by 30 cycles of 30 s at 94uC, 30 s at 55uC and 60 s at 72uC. A final elongation step of 10 min at 72uC was included. Figure 7. Quantitative assessment of the silencing efficiency of ASON-Bulge-DNAzyme against luciferase. BHK21 cells were harvested and lysed by in 200 ml lysis buffer (0.1 M Tris-HCl, 0.1% Triton X-100, 2 mM EDTA, pH 7.8). The luciferase activity was measured using a luminometer. The error bars denote Standard deviation (SD). doi:10.1371/journal.pone.0018629.g007 Semi-quantitative RT-PCR analysis of integrin genes Two pairs of specific primers were designed according to the sequence of the integrin b1 gene and the integrin b3 gene. The primers were synthesized by Shanghai Generay Biotech Co., Ltd. Integrin b1-F1: 59-ATCCCAGAGGCTCCAAAGATAT-39 Integrin b1-R1: 59-CACCAAGTTTCCCATCTCCAG-39 Integrin b3-F1: 59-AGGATTACCCTGTGGACATC-39 Integrin b3-R1: 59-CCGTGATCTTGCCAAAGTCAC-39 cDNA was synthesized using 4 ml of the total RNA, 4 ml of 56reverse transcriptase buffer, 1 ml of 10 mM dNTPs, 1 ml of integrin b1-R1 primer (10 mM), 1 ml of integrin b3-R1 primer (10 mM), 1 ml of RNase inhibitor (20 U/ml), 1 ml of MMLV reverse transcriptase (5 U/ml) and 6 ml of RNase-free H2O in a total volume of 20 ml for 1 h at 42uC. The PCR conditions were as follows: 1 cycle at 94uC for 5 min; followed by 35 cycles of 40 s at 94uC, 30 s at 55uC and 60 s at 72uC. A final elongation step of 10 min at 72uC was included. Cell culture and transfection BHK-21 cells (from ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, USA) containing 10% fetal bovine serum (FBS) supplemented with 1% penicillin– streptomycin (GIBCO). Cells were cultured in 24-well plates and transfected by Lipofectamine 2000 (Invitrogen, USA). The ratio of DNAzyme to Lipofectamine was 1 mg:2 ml. Cells were co- transfected with 0.5 mg of pECFP-C1 plasmid and 0.25 mg of siRNA expression cassettes in each well. Western blot analysis BHK-21 cells were harvested 36 h after eGFP DNAzyme transfection. They were then washed with PBS three times, and lysed with 100 ml of lysis buffer. Total cellular protein was determined using the BioRad Protein Assay reagent (Bio-Rad Laboratories). The eGFP was detected using mouse anti-GFP PLoS ONE | www.plosone.org 5 April 2011 | Volume 6 | Issue 4 | e18629