Applications: RNAi Gene Silencing

shRNA Mediated Gene Silencing

Short hairpin RNA (shRNA) mediated gene silencing is a widely used approach for stable gene knockdown; these short RNA sequences can be expressed via viral or non-viral DNA vectors that encode shRNA.

While shRNA mediated silencing can be employed to generate stable knockdown cell lines, this approach can be time-consuming. Additionally, cell types such as primary cells may yield low transfection efficiencies through shRNA plasmid based transfection as opposed to cytoplasmic siRNA delivery.

A common vehicle for shRNAs is viral transduction through:

• Adeno-associated virus (AAV) or adenovirus - Expression through AAV or adenovirus can prevent insertional mutagenesis since these vectors remain episomal, but this approach leads to more transient expression since the vectors are lost through multiple rounds of cell division
• Lentivirus - Expression through lentivirus provides a stable solution through chromosomal integration, but this also presents the risk of insertional mutagenesis

Large scale shRNA libraries are increasingly becoming available to enable RNAi research. For hard-to-transfect cells including primary cells, generating stable cell transfectants via lentivirus or retrovirus transduction is a viable alternative. In this case, antibiotic resistance harboring virus particles generated after transfection of producer cell types such as HEK293T are used to transduce cells that can then be selected for virus integration. Details on virus production can be found here. A popularly used protocol for preparing lentiviral shRNA vectors can be found at the public TRC portal protocol database of the RNAi Consortium at the Broad Institute.

TransIT-X2® Dynamic Delivery System is the reagent of choice for delivering plasmid DNA encoding shRNA. TransIT®-2020 and TransIT®-LT1 Transfection Reagents can also effectively transfect plasmid DNA encoding shRNA into mammalian cells in culture. Ideally, all three reagents should be tested in parallel to determine the best solution for your application. Depending on the cell type, one reagent may have superior performance over the others. For cell-type specific recommendations, please consult the Reagent Agent® transfection database.

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For more information on transfection for RNAi applications, additional resources can be found on the Mirus Bio website:

Optimize siRNA Transfection
Deliver microRNA (miRNA) Effectively
Reverse Transfection Protocol for siRNA/miRNA