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The Thermo Scientific GIPZ microRNA-adapted shRNA collection was developed in collaboration with Gregory Hannon and Steve Elledge at Cold Spring Harbor Laboratories (CSHL). GIPZ shRNA designs are based on native miR-30 primary transcript to enable processing by the endogenous RNAi pathway and result in specific gene silencing with minimized cellular toxicity.
*One out of three GIPZ Lentiviral shRNA is guaranteed to produce gene knockdown by ≥ 70% at the mRNA level when used in shRNA Starter Kit. Knockdown should be compared to non-silencing control and experimental workflow must be controlled using the provided GIPZ GAPDH positive control.
GIPZ Lentiviral shRNA Starter Kits
The Thermo Scientific GIPZ Lentiviral shRNA Starter Kits combine the versatility of lentiviral shRNA, the convenience of experimental controls and the necessary mode of delivery for RNAi experimentation.
The GIPZ Transduction Starter Kit includes:
The GIPZ Transfection Starter Kit includes:
***Please note that due to the inherent complexity of high-titer lentiviral particle production, we estimate a 6 to 8 week turnaround time.
† When you purchase a minimum of three shRNA to the same target, at least one of the shRNA constructs will reduce target mRNA levels by 70% or more when used with Starter Kit protocols and normalized for transfection efficiency. Transfection conditions should be confirmed using appropriate positive controls provided in the kit and the percent-knockdown should be compared to cells transfected with the negative control (non-targeting) shRNA.
‡ For a list of positive controls included in each Starter Kit, please see the relevant specification sheet.
Please note that the GIPZ Lentiviral shRNA and TRIPZ Inducible Lentiviral shRNA are not compatible with third generation packaging systems, such as ViraPower from Invitrogen. We recommend the Trans-Lentiviral Packaging System to general lentiviral particles for transduction of GIPZ and TRIPZ shRNA.
Figure 2. OVCAR-8 cells were transduced with GIPZ lentiviral shRNA constructs at MOI = 0.4 to2 in 2 to 4 biological replicates. Cells were puromycin-selected (30 μg/mL) starting 48 hours post-transduction. RNA was isolated 84 hours posttransduction.
qPCR was performed in triplicate via TaqMan® Gene Expression Assays using 18S rRNA as an internal reference. On average, two out of three shRNA produced greater than 70% knockdown compared to the GIPZ Non-targeting Control shRNA.
Figure 3. HEK293T cells were transduced with lentiviral particles containing shRNA to GAPDH, eG5, p53, Rb, or STAT6. Non-silencing shRNA was transduced as a control. The graph depicts the residual levels of these genes normalized to their equivalent non-silencing control.
Figure 4. TurboGFP expression from the pGIPZ vector allows measurment of transfection and transduction efficiency. It also effectively marks shRNA expression in target cells. (A) HEK293 cells 48 hrs post-transduction and (B) the corresponding phase image.