MISSION® shRNA Controls

When conducting experiments using MISSION TRC shRNA constructs, proper controls are a key element of experimental design to permit accurate interpretation of knockdown results and provide assurance of the specificity of the response observed. Sigma® offers a variety of positive and negative controls to monitor for efficient transduction, knockdown, and off-target effects.

All controls are available in both purified plasmid DNA and lentiviral particle format. The DNA format controls may be used in direct transfection-based experiments or may be used in conjunction with the MISSION Lentiviral Packaging Mix (SHP001) to create replication incompetent viral particles for use in transduction-based experiments.

Sigma's recommended controls for any shRNA experiment are provided in this Control Selection table and are closely aligned with the controls suggested in the Nature Cell Biology editorial.¹

Recommended Control	Objective
Negative Control: Untreated Cells	Untreated cells will provide a reference point for comparing all other samples.
Negative Control: Empty construct, containing no shRNA insert (SHC001V or SHC001)	MISSION pLKO.1-puro Control The empty viral particles or DNA are a useful negative control that will not activate the RNAi pathway because it does not contain an shRNA insert. It will allow for observation of cellular effects of the transduction/transfection process. Cells transduced/transfected with the empty control provide a useful reference point for comparing specific knockdown.
Negative Control: Non-targeting shRNA (SHC002V or SHC002)	MISSION Non-Target shRNA Control This non-targeting shRNA is a useful negative control that will activate RISC and the RNAi pathway, but does not target any human or mouse genes. The short hairpin sequence cotnains 5 base pair mismatches to any known human or mouse gene. This allows for examination of the effects of shRNA transduction/transfection on gene expression. Cells transduced/transfected with the non-target shRNA will also provide useful reference for interpretation of knockdown.
Positive Control: Positive reporter vector or lentiviral particles (SHC003V or SHC003)	MISSION TurboGFP Control This is a useful positive control for measuring transduction/transfection efficiency and optimizing shRNA delivery. The TurboGFP Control contains a gene encoding TurboGFP, driven by the CMV promoter. This control provides fast visual confirmation of successful transduction/transfection. Cells expressing TurboGFP 48 hours post-transduction. Lentiviral titer was estimated to be 1.0 x 106 TU/ml using a p24 based ELISA.
Positive Control: Positive shRNA knockdown control (SHC004V or SHC004)	MISSION TurboGFP shRNA Control This control contains shRNA sequence that targets TurboGFP expression. The shRNA has been experimentally shown to reduce TurboGFP expression by 99.6% in HEK293T cells 24 hours post-transduction. This control serves to quickly visualize knockdown in cells expressing TurboGFP.
Positive Control: Positive shRNA knockdown control (SHC005V or SHC005)	MISSION eGFP shRNA Control This control contains shRNA sequence that targets eGFP expression (GenBank Accession # pEGFP U476561). The shRNA has been experimentally shown to reduce eGFP expression by 90% in C166-GFP mouse fibroblast cells 48 hours post-transduction by mRNA transcript level. This control serves to quickly visualize knockdown in cells expressing eGFP. Knockdown efficiency of eGFP shRNA in HEK293T cells. Images shown 48 hours post-transfection. A) 100 ng of eGFP expression plasmid alone. B) 100 ng eGFP plasmid + 200 ng MISSION eGFP shRNA Control Vector. C) Bright field illumination.
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Reference

1. Whither RNAi? Nature Cell Biology, 5, 489-490 (2003).

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MISSION is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. Label License.

TurboGFP is a trademark of Evrogen Co.

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