Validated shRNA controls

• Changes in the mRNA or protein levels in cells treated with negative or non-silencing controls reflect non-specific responses in cells and can be used as a baseline against which specific knockdown can be measured.
• Positive controls are useful to demonstrate that your experimental system is functional and your shRNA construct is successfully activating the RNAi pathway.
• Knowledge of transfection efficiency in gene knockdown experiments is also critical in understanding the results. Factors affecting transfection efficiency include purity of plasmid DNA, health of transfected cells, inconsistencies in number of cells plated, insufficient mixing of transfection complexes, different cell types or change in the cell passage number. Determining the transfection efficiency for every RNAi experiment is thus essential to enable valid comparisons across sets of data.

Available Expression Arrest shRNAmir controls:

• eGFP shRNAmir construct: The eGFP shRNA is a positive control designed against the enhanced GFP reporter (GenBank Accession No: pEGFP U476561 ). This construct* has been validated to produce > 75% decrease in GFP fluorescence. The eGFP shRNA sequence has been cloned into pSM2 retroviral and the pCMV-GIN-ZEO lentiviral vectors.

Validating controls for RNAi

The data is shown as percent suppression of luciferase activity normalized to the non-silencing control. The firefly luciferase shRNA decreased luciferase activity by nearly 80% relative to the non-silencing control whereas the eGFP shRNA used as a negative control did not suppress activity. The data is shown as eGFP activity normalized to the non-silencing control. The eGFP shRNA illustrated a high level of silencing relative to the non-silencing shRNA whereas the Luciferase shRNA as a negative control did not affect the activity.