Positive and negative controls are a critical part of any gene silencing experiment as they enable accurate interpretation of knockdown results and provide confidence in the specificity of the response. GIPZ positive and negative shRNA controls are available as E. coli glycerol stock clones or high-titer lentiviral particles.
- All GIPZ shRNA constructs include the TurboGFP reporter gene for convenient tracking of expression and puromycin-resistance for mammalian cell selection.
- Changes in the mRNA or protein levels in cells treated with a GIPZ negative (non-targeting or non-silencing) shRNA control reflect non-specific responses in cells and should be used as a baseline against which specific knockdown can be measured.
- A GIPZ positive shRNA control targeting GAPDH or EG5 will confirm whether the construct is successfully delivered into the cell and can activate the RNAi pathway.
Controls can also facilitate understanding of transfection efficiency of your cells of interest. 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 shRNA Controls Include
- Non-silencing shRNA construct - The non-silencing shRNA is a negative control for any transfection experiment performed using GIPZ shRNA constructs. When transfected into cells, the endogenous RNAi pathway processes it, but its processed siRNA will not target any mRNA in the mammalian genome. The non-silencing shRNA sequence is cloned into GIPZ lentiviral and TRIPZ lentiviral vectors. This sequence is 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 shRNA library include shRNA targeting EG5 (human) and GAPDH (human and mouse). EG5 and GAPDH shRNA containing vectors are available in glycerol stock or viral particle formats. The GAPDH control is available in the TRIPZ vector.
- 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:
- β -gal reporter - Monitors transfection efficiencies across wells and under different experimental conditions
Glycerol stocks of shRNA containing control vectors are shipped as bacterial cultures of E.coli in LB (low salt) broth with 8% glycerol, carbenicillin (100 µ/mL) and zeocin (25 µ/ml). We check all cultures for growth prior to shipment.
Viral particle formats are provided as frozen stocks containing 10^7-10^8 transducing units per mL in DMEM (Dulbecco's Modified Eagle's Medium) with no serum or antibiotics. All shRNA constructs should be stored at -80°C.
Designed by Dr Ricky Rickles, Harvard Medical School.
- Editorial, Whither RNAi? Nature Cell Biology.5(6), 489-490 (2003) .
pGIPZ lentiviral vector elements