Controls are a critical part of a gene silencing experiment. They enable accurate interpretation of knockdown data and provide confidence in the specificity of the response. Open Biosystems have developed and validated positive and negative controls for shRNAmir libraries. These are available as glycerol stock or viral particle (for GIPZ lentiviral controls) formats.
- 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.
Recommended controls for RNAi studies are based on suggested controls detailed in an editorial published in Nature Cell Biology1
Available shRNAmir controls include:
•
Non-silencing shRNAmir construct -The non-silencing shRNAmir is a negative control for any transfection experiment performed using GIPZ or pSM2 shRNAmir constructs. When transfected into cells it will be processed by the endogenous RNAi pathway but its processed siRNA will not target any mRNA in the mammalian genome. The non-silencing shRNAmir sequence has been cloned into
pSM2 retroviral,
pSMP retroviral,
GIPZ lentiviral, and
TRIPZ lentiviral vectors. This sequence has been verified to contain no homology to known mammalian genes. Both glycerol stock and viral particle formats are available.
•
Endogenous positive controls for the
GIPZ lentiviral shRNAmir library include shRNAmir targeting EG5 (human) and GAPDH (human and mouse). EG5 and GAPDH shRNAmir containing vectors are available in glycerol stock or viral particle formats. The GAPDH control is available in the
pSMP and
TRIPZ vector as well.
• Exogenous positive controls are a good choice as positive controls. They control for expression level of your gene in the target cell line and abundance and turnover rate of the target mRNA/protein. Available exogenous positive controls include:
• eGFP shRNAmir construct: The eGFP shRNA is a positive control designed against the enhanced GFP reporter (GenBank Accession No: pEGFP U76561 ). This construct* has been validated to produce > 75% decrease in GFP fluorescence. The eGFP shRNA sequence has been cloned into pSM2 retroviral vector.
• Luciferase shRNAmir construct: The Firefly Luciferase shRNA is a positive control designed against pGL3 Firefly Luciferase (Promega Cat. #E1741). This construct has been validated to produce >75% decrease in luciferase activity. The luciferase shRNA sequence is cloned into pSM2 retroviral vector.
• β -gal reporter - Monitors transfection efficiencies across wells and under different experimental conditions
