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Applications | RNAi Gene Silencing

TransIT® Transfection Reagents for Gene Silencing

Illustration highlighting siRNA, shRNA and miRNA mediated pathways

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 TransfectedTransfection Reagent Recommendation
siRNA/miRNAOption 1: TransIT-X2® Dynamic Delivery System
Option 2: TransIT-TKO® Transfection Reagent
Option 3: TransIT-siQUEST® Transfection Reagent
shRNA encoding Plasmid DNAOption 1: TransIT-X2® Dynamic Delivery System
Option 2: TransIT®-2020 Transfection Reagent
Option 3: TransIT®-LT1 Transfection Reagent
siRNA/miRNA and Plasmid DNATransIT-X2® Dynamic Delivery System

siRNA Mediated Gene Silencing

 

siRNA mediated pathway illustration

Apart from their natural occurrence, exogenous sequences of siRNAs can be designed and introduced into cells through transfection to knock down relevant gene expression. This application serves as a tool to elucidate genetic pathways, determine protein function, or uncover new gene targets for biotherapeutic and pharmaceutical applications. Larger libraries of these siRNA molecules are also available to perform larger genome RNAi analysis via high-throughput RNAi screening.

Exogenous sequences of siRNAs… can be designed and introduced into cells through transfection to knock down relevant gene expression.

Because siRNA differs in size and structure from plasmid DNA, transfection reagents can be optimized and formulated separately for delivery of these RNA molecules. In addition, delivery of siRNA to the cytoplasm for incorporation into the RISC complex is sufficient for gene knockdown. Selection of the appropriate transfection methodology or reagent must first be considered, followed by further optimization for efficient siRNA delivery and subsequent gene knockdown. Optimization of several experimental parameters is key to achieving the highest efficiency siRNA transfection and corresponding effective knockdown of target gene expression. 

A high and reliable level of knockdown can be achieved through siRNA transfection via a variety of quality transfection reagents and validated siRNA libraries. However, a few disadvantages with siRNA mediated knockdown are the chances of off-target effects and transient knockdown through siRNA dilution after multiple cell divisions. A viable alternative in this scenario is shRNA mediated gene-silencing.

If direct tracking of siRNA delivery is desired, siRNAs can also be labeled with different fluorophores using Label IT® siRNA Tracker™ Intracellular Localization Kits. See figure below.

TransIT-X2® Dynamic Delivery System is the reagent of choice for delivering siRNA. TransIT-X2® delivers both plasmid DNA and/or siRNA efficiently and can be used for co-transfecting plasmid DNA and siRNA. TransIT-TKO® and TransIT-siQUEST® Transfection Reagents can also effectively transfect siRNAs 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.

TransIT-TKO Transfection Reagent Successful Delivery and Functional Knockdown Data

 

Successful Delivery and Functional Knockdown Using Labeled siRNA. (A) HeLa cells in 12-well plates were transfected at 70% confluence with TransIT-TKO® Transfection Reagent (3 μl/well) and Label IT® siRNA Tracker™ Fluorescein-labeled siRNA duplexes (GREEN, 50 nM final concentration in the well). The cells were incubated 24 hours post-transfection then fixed and counterstained with TO-PRO®-3 (nuclei, BLUE) and Alexa Fluor® 546 Phalloidin (actin, RED). Confocal images were acquired on a Zeiss LSM 510 Confocal Microscope. (B) TransIT-TKO® Transfection Reagent was used to transfect anti-firefly luciferase siRNA into CHO-Luc cells stably expressing firefly luciferase. The siRNA was either unlabeled or labeled with Label IT® siRNA Tracker™ Cy®3, Cy®5, Fluorescein, or CX-Rhodamine Reagents. Bars indicate the percent firefly luciferase expression 24 hours after delivery of 5 nM anti-firefly luciferase siRNA.

miRNA Mediated Gene SilencingmiRNA mediated pathway illustration

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

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.

 

TransIT-X2 Dynamic Delivery System miRNA delivery Data

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.

shRNA Mediated Gene SilencingshRNA mediated pathway illustration

 

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.
 
 
TransIT Transfection Reagent Protocol schematic

TransIT® Transfection Reagents are Ideal for Virus Production. All TransIT® Transfection Reagents are low toxicity and do not require a media change. Save time and money by adding formed complexes directly to cells in media containing serum and avoiding unnecessary media changes.

Products for Gene Silencing

The following transfection solutions are available to researchers employing gene siliencing in their experiments. All TransIT® transfection reagents can be used for gene silencing with the following general recommendations based on the nucleic acid to be delivered. 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 TransfectedTransfection 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 libraries

Option 1: TransIT-X2® Dynamic Delivery System

Option 2: TransIT®-2020 Transfection Reagent

Option 3: TransIT®-LT1 Transfection Reagent

siRNA/miRNA and Plasmid DNATransIT-X2® Dynamic Delivery System
 

 


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