The SMARTchoice Inducible shRNA experimental workflow begins with using the SMARTchoice Inducible Non-targeting Control 4-Pack to identify the most active vector configuration in your cells of interest simply by assessing TurboGFP fluorescence intensity in transduced cells. Then, order gene-specific shRNAs and matched-vector positive and negative controls with your choice of RNA polymerase II promoter and reporter. Optimization of doxycycline doses (concentrations) and time points for induction should be performed using positive and negative controls. Transduce cells with SMARTchoice Inducible shRNAs targeting genes of interest, always including matched positive and negative RNAi controls for rigorous data interpretation. All SMARTchoice Inducible shRNAs and RNAi controls are provided as high-titer, purified and concentrated lentiviral particles for immediate transduction.
SMARTchoice Inducible Non-targeting Controls were used to transduce the indicated cell types at MOI = 0.3. 24 h after transduction, expression of the non-targeting shRNA and PuroR was induced with 1 µg/mL doxycycline. After 48 h of culture in the presence of doxycycline, cells were stained with Hoescht-33342 and nuclei (blue) and TurboGFP (green) were imaged. While ~30% of the cells in the field have been transduced, some images may appear to contain fewer than 30% TurboGFP-positive cells due to low TurboGFP expression, indicative of low constitutive promoter activity, in a particular cell type.
A. U2OS cells were transduced at MOI = 0.1 with SMARTchoice Inducible mCMV vectors carrying either a non-targeting control shRNA (NTC) or an shRNA directed against the ubiquitin B (UBB) gene. Cells were selected for 3 days with 1.5 µg/mL puromycin. After selection, cells were seeded at 2000 cells per/well in 96-well plates. 24 hours later (Day 1), shRNA expression was induced with the indicated dose of doxycycline, and cell number was then measured each day with the Cell Titer-Glo® assay (Promega). Each data point represents the mean and standard deviation of six independent wells. B. On Day 6, following 5 days of exposure to doxycycline, the cells were stained with Hoescht 33342 and cell nuclei (blue) and TurboGFP (green) were imaged on the ArrayScan™ VTI HCS Reader. C. Cell density, defined as number of nuclei per field was computed for 18 fields per condition.