miRNA Mediated Gene Silencing

Similar to siRNAs, exogenous sequences of miRNA related small RNAs can be designed and introduced into cells through transfection to knock down relevant gene expression.

Exogenous Sequences of miRNA mimics, pre-miRNAs and miRNA Inhibitors can be Designed and Introduced into Cells through Transfection to Investigate miRNA Function

The most popular classes of miRNA related RNA molecules that are transfected to investigate gene function are miRNA mimics, pre-miRNAs and miRNA inhibitors.

  • miRNA mimics are chemically synthesized miRNAs that mock miRNAs occurring in nature
  • Pre-miRNAs are larger nuclear precursors of miRNA that can range in size from ~60-70 bases and are transported to the cytoplasm for cleavage into mature miRNA
  • miRNA inhibitors are single-stranded, chemically modified RNAs that incorporate reverse complements of miRNA target sequence and thereby inhibit miRNA function
miRNA

TransIT-X2® Dynamic Delivery System is the reagent of choice for delivering miRNA, miRNA mimics or pre-miRNAs (see figure below). For miRNA transfection, follow the user protocol and simply substitute duplex miRNA for siRNA. TransIT-X2® delivers both plasmid DNA and/or miRNA efficiently and can be used for co-transfecting plasmid DNA and miRNA.

TransIT-TKO® and TransIT-siQUEST® Transfection Reagents can also effectively transfect duplex miRNAs 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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Effective miRNA Delivery using TransIT-X2 Dynamic Delivery System Yields Decreased Levels of PTK9 mRNA

Effective miRNA Delivery using TransIT-X2® Dynamic Delivery System Yields Decreased Levels of PTK9 mRNA. TransIT-X2® Dynamic Delivery System and Lipofectamine® 2000 Transfection Reagent were used to transfect Pre-miR™ hsa-miR-1 miRNA Precursor or mirVana™ miRNA mimic, miR-1, both known to decrease PTK9 mRNA levels. A Pre-miR™ negative control was transfected to assess baseline mRNA levels. T47D cells were transfected in a 12-well plate using 3 µl of TransIT-X2® or Lipofectamine® 2000 and 50 nM miRNA according to each manufacturer's protocol. The amount of PTK9 mRNA was measured relative to 18s rRNA levels using qRT-PCR and then normalized to the mRNA levels of the negative control, 48 hours post-transfection. Error bars represent the standard deviation of triplicate wells.


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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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