siARRAY™ Reverse Transfection Format (RTF):
A Rapid Method for RNAi-based High Throughput
Studies of Biological Pathways

RNA interference (RNAi) screens are easier and more efficient with the Dharmacon siARRAY RTF™ small interfering RNA (siRNA) Libraries, which combine optimized, broad-spectrum transfection reagents and potent SMARTpool® siRNA into simple, assay-ready and automation-compatible formats.

Queta Boese, PhD

The availability of siRNA collections targeting related genes offers the possibility of high throughput studies; however, managing large-scale transfection of siRNA reagents is time consuming and costly. To facilitate high throughput analyses, Dharmacon has developed a strategy to streamline the transfection workflow, permitting rapid and economical screening. The siARRAY RTF siRNA Libraries contain preselected groups of rationally-designed SMARTpool siRNA reagents targeting genes confirmed to be relevant to a particular pathway or phylogenetically related to the indicated gene family as defined by the Gene Ontology Consortium (http://www.geneontology.org). 

The distinguishing feature of siARRAY RTF is that the SMARTpool reagents are provided in a pre-aliquoted format, dried down in 96-well plates, such that cells are plated simultaneously into wells containing the individual SMARTpool silencing reagents rehydrated with a lipid/media mixture.

Here we illustrate this novel transfection strategy in the functional analysis of targets implicated in clathrin-mediated endocytosis (CME) demonstrating siARRAY RTF siRNA Libraries as invaluable tools for robust and reliable high throughput screens.


Delivery– Conventional Transfection vs. Reverse Transfection Formats

Conventional transfection, also known as forward transfection (FT), is the most common technique for delivering siRNA into cells for gene silencing.  This standard approach involves pre-plating cells approximately one day prior to treatment.  On day two, siRNA designed to target a specific gene is complexed with a transfection reagent suitable for delivery into the cells.  Silencing is then assessed 24 to 48 hours later (or more) depending on the target and the detection method. For multiple targets or high throughput strategies, FT requires significant handling of individual samples, such that screens with hundreds or thousands of genes quickly become laborious and cost-prohibitive.

To overcome this challenge, Dharmacon developed a new reverse transfection format (RTF). RTF differs from FT by virtue of streamlined transfection preparation and set-up (Figure 1).  The SMARTpool reagents are pre-aliquoted as a siARRAY RTF Library in 96-well plates, supplied in triplicate, and ready for a one-time transfection of 50 µM siRNA into the cells of interest.  Each plate also contains three negative and three positive control siRNAs and is shipped complete with DharmaFECT™ Transfection Reagent and DharmaFECT Cell Culture Reagent (DCCR).  Following a very simple protocol, the SMARTpool reagents are rehydrated in each well with a mixture of DCCR and DharmaFECT. After a short incubation period (30 to 90 minutes), cells are added to each well and incubated under standard conditions. Silencing is then assessed using an assay appropriate for the pathway of interest. 

Under the conditions just described, RTF achieves efficient and specific target knockdown with minimal effect on cell viability, and at lower overall concentrations of transfection reagent and siRNA relative to FT.  By eliminating complex and time-consuming manual processing, siARRAY RTF reduces handling to merely rehydrating the pre-aliquoted SMARTpool siRNAs and adding the cells.  Thus, siARRAY RTF Libraries minimize the potential for exposure to ribonucleases and provide a means for reliable and robust targeted gene knockdown. 

Application & Experimental Format

To demonstrate the utility of the RTF approach, it was compared to FT in a functional analysis of genes involved in the clathrin-mediated endocytosis (CME) pathway.  CME is an important process in higher eukaryotes for the internalization of nutrients, macromolecules, viruses, and plasma membrane proteins from the extracellular environment [4] and is of interest to those studying host-viral pathogen interactions. 

In previous work, thirteen genes (CHC, b2-adaptin, dynamin II, CALM, Eps15, Eps15R, Epsin, EEA.1, Rab5a, Rab5b, Rab5c, CLCa, and CLCb) were targeted in HeLa cells by SMARTpool reagents using conventional FT.  These targets were chosen based on their purported involvement in CME and the availability of antibodies for detection [5]. The consequence of RNAi-mediated gene knockdown was assessed by Western blot analysis, and - where antibodies were lacking - by monitoring expression levels of yellow fluorescent protein (YFP) -tagged constructs.  The effect of targeted gene knockdown was also assessed quantitatively by monitoring the internalization of ¹²⁵I-Transferrin (Tfn) (Figure 1a).  Tfn is a ligand that, upon binding to its receptor, is endocytosed constitutively through clathrin-coated pits. In all cases, the corresponding SMARTpool reagent and at least one of the individual siRNAs reduced protein levels by >90% (all SMARTpool reagents and at least three of four individual siRNAs reduced protein levels by 75%) [5]. 

It was determined that the analysis of an expanded set of 44 genes involved in CME (Table 1) represented a suitable test case for the feasibility of RTF-based delivery and silencing.  The corresponding siARRAY RTF collection was rehydrated according to the included protocol.  HeLa cells were then plated at a density of 15,000 cells/well.  Uptake of fluorescein isothiocyanate-labeled Tfn (FITC-Tfn) was assessed in a short time course assay at 37°C to avoid the contribution of recycled ligand. The resulting phenotypes, determined by the presence or absence of labeled vesicles (endosomes) and labeled plasma membrane, were scored by visualization of FITC-Tfn internalization  (Figure 1b).  For example, RNAi-mediated silencing of Eps15 had no effect on the endocytosis of FITC-Tfn while knockdown of CHC, b2-adaptin, and the combined knockdown of Rab5a, Rab5b and Rab5C resulted in strong visual phenotypes (Figure 1b).  These results were consistent with those of FT, demonstrating the reproducibility of siARRAY RTF Libraries in rapid screens of large gene families.


Conclusions

The application of RNAi-mediated silencing in high-throughput functional analyses can be a laborious endeavor by virtue of the number of samples to be processed. siARRAY RTF siRNA Libraries represent an important advancement in critical screening tools that combine optimized broad-spectrum transfection reagents and potent SMARTpool siRNAs into simple, assay-ready, and automation-compatible formats.  Depending on the screening assay, a screen of hundreds of targets could be completed within a matter of days. 

As described here, siARRAY RTF application distinguishes itself by permitting efficient transfection and rapid screens. The functional analysis of genes involved in CME was consistent with that achieved by FT, thus RTF provides a cost-effective strategy for quick, reliable high throughput screening and target validation methods.

Links
Dharmacon Inc. (www.dharmacon.com)
Gene Ontology Consortium (http://www.geneontology.org/)

References

Figures and Tables

Figure 1. Workflow comparison for Forward Transfection and Reverse Transfection formats illustrating the labor and reagent-savings features.

Figure 1a (bar graph). RNAi-mediated silencing via FT of specific CME proteins individually (e.g., CHC, beta2) or in combination (e.g., Rab5a, Rab5b, and Rab5c) affect internalization of ¹²⁵I-Transferrin (Tfn) [5].

Figure 1b (cell photos). RTF recapitulates phenotypes observed by FT of siRNAs targeting a collection of proteins involved in CME.  Fluorescent img of FITC-Transferrin internalization in HeLa cells treated with target specific siRNAs.  [Symbol 1 – Eps15 knockdown has no effect ; Symbol 2 - CHC knockdown results in a strong phenotype]