TransIT® Transfection Reagents for Gene Silencing

The ability to silence genes plays an important role in molecular and cell biology and can be readily applied through transfection. Gene expression can be effectively reduced or eliminated by introducing small noncoding RNA molecules that inhibit RNA translation though a process termed RNA interference (RNAi). RNAi-based approaches rely on the inherent cellular machinery, shared among several eukaryotic organisms, to inhibit mRNA translation. RNAi pathways are believed to provide a mechanism to protect cells from extraneous nucleotide sequences (e.g., viruses and transposons) but, more recently, exhibited an important role in regulating gene expression.

RNA molecules that take part in naturally occurring RNAi pathways include:

  • Small interfering RNAs (siRNA) - short (20-25 base pairs) double-stranded RNAs resulting from the cleavage of double-stranded RNAs (dsRNAs)
  • MicroRNAs (miRNAs) - a separate class of short single-stranded RNAs (20-22 nucleotides) resulting from the processing of noncoding RNAs

Both of these RNAi pathways rely on cellular machinery such as the ribonuclease protein DICER and the RNA-induced silencing complex (RISC). DICER initiates the RNAi pathway by processing dsRNA to form siRNAs or mature miRNAs. These RNA molecules can bind to complementary sequences of mRNA within the RISC, and the mRNA can be cleaved by the catalytic component, Argonaute, which ultimately prevents translation.

An alternative approach that takes advantage of RNAi to knock down gene expression is the use of short hairpin RNA (shRNA). These short RNA sequences can be expressed via viral or non-viral vectors. shRNA expression mimics a pathway similar to siRNA/miRNA since the expression product must be processed by DICER and ultimately incorporated into the RISC complex for targeted degradation of mRNA.

Whether shRNA, siRNA or miRNA mediated RNAi approaches are implemented, the overall goal of the gene silencing experiment needs to be established. One must also consider the gene targets and cell types used, design the proper sequence for specificity, determine duration of expression and select the most effective means of small RNA delivery to ensure success.

The following transfection solutions are available to researchers employing gene siliencing in their experiments. All of the TransIT® siRNA/miRNA transfection reagents can be used for gene silencing with the following general recommendations based on the nucleic acid to be delivered. All of these broad spectrum formulations are chemically distinct. Depending on the cell type being transfected, one reagent may have superior performance over others; for cell-type specific recommendations, please consult the Reagent Agent® transfection database.

Broad Spectrum TransIT® Transfection Reagent Recommendations for Gene Silencing

Nucleic Acid to be Transfected Transfection Reagent Recommendation
siRNA/miRNA Option 1: TransIT-X2® Dynamic Delivery System
Option 2: TransIT-TKO® Transfection Reagent
Option 3: TransIT-siQUEST® Transfection Reagent
shRNA encoding Plasmid DNA Option 1: TransIT-X2® Dynamic Delivery System
Option 2: TransIT®-2020 Transfection Reagent
Option 3: TransIT®-LT1 Transfection Reagent
siRNA/miRNA and Plasmid DNA TransIT-X2® Dynamic Delivery System
 

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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

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