A variety of siRNA applications, including siRNA library screening, require the efficient delivery of hundreds to thousands of siRNAs to cells. Most published siRNA studies describe performing transfections in 6 well and 24 well formats. Ambion has developed two reagents, siPORT NeoFX Transfection Agent and siPORT siRNA Electroporation Buffer, and an apparatus, the siPORTer-96 Electroporation Chamber, to facilitate siRNA delivery in 96 well formats. Here we describe experiments analyzing the efficiency and reproducibility of these tools for delivering siRNAs using standard laboratory equipment.
Chemical transfection and electroporation are routinely used to deliver siRNAs into cultured cells. Of these two methods, chemical transfection is most effective for delivering siRNAs into adherent immortalized cell lines, whereas electroporation is recommended for primary cells , cells grown in suspension, and other chemical transfection-resistant cell lines. We have adapted both methods for high throughput delivery of siRNAs.
High Throughput Delivery
High throughput siRNA delivery remains a challenge for many laboratories due to the variability of cell culture and delivery efficiency in 96 and 384 well plate formats. There are three important components of a successful siRNA library screen that are directly related to delivery:
Transfection/electroporation effici uct Name Size 4510 siPORT NeoFX Transfection Agent 0.4 ml 4511 siPORT NeoFX Transfection Agent 1 ml 4605 Silencer GAPDH siRNA (Human) 5 nmol + 2 nmol Neg Control (50M) 4624 Silencer GAPDH siRNA (Human, Mouse, Rat) 5 nmol + 2 nmol Neg Control (50M) 8990 siPORT siRNA Electroporation Buffer 12 x 1.5 ml 13500 siPORTer-96 Electroporation Chamber 1 device ency maximizes reduction in target gene expression and improves the likelihood that a significant phenotype will be observed.
Reproducibility of siRNA delivery between wells and between plates reduces the variability of quantitative phenotypic results and improves the resolution of screening data.
Cell viability increases specificity of the siRNA effect, further improving the likelihood that significant phenotypic differences will be observed.
High Throughput Chemical Reverse Transfection--For Immortalized Cells
We adapted reverse transfection (a variation of standard transfection that involves transfection in suspension; see "Reverse Transfection" at right) to facilitate high throughput siRNA delivery. Because the procedure features minimal pipetting steps and can be performed using multi-channel pipettors, reverse transfection can be used to deliver siRNAs to approximately 1000 cell samples per hour without robotics. It is also amenable to automation
Efficiency of High Throughput Reverse Transfection. To assess transfection efficiency, we mixed different concentrations of a well-characterized GAPDH siRNA with the versatile siPORT NeoFX Transfection Agent in wells of a 96 well plate. Five different cell types were added to the siRNA/lipid complexes to effect reverse transfection. 48 hours later, GAPDH mRNA levels were monitored by real-time RT-PCR (Figure 1A). For comparison, GAPDH siRNA was delivered by a standard transfection protocol (pre-plated) in two of the cell types (Figure 1B).
Figure 1. Efficiency of Reverse Transfection in Multiple Cell Types. (A) Indicated cell lines were plated in 96 well format (8000 cells/well) and simultaneously transfected by adding transfection complexes, prepared in Opti-mem serum-free medium (Life Technologies) by mixing 0.3 l siPORT NeoFX Transfection Reagent (Ambion) and 1 nM siRNA targeting GAPDH (Silencer GAPDH siRNA, Ambion) or non-silencing control (Silencer Negative Control #1 siRNA, Ambion). 48 hours post-transfection cells were harvested and analyzed by real-time RT-PCR for both target mRNA and 18S rRNA levels. Remaining gene expression was calculated as a percentage of target mRNA in cells transfected with siRNA targeting GAPDH and cells transfected with the non-silencing control siRNA. Data was normalized against the 18S rRNA signal. Transfections were performed in duplicate. Data are presented as means SD. (B) HepG2 and HeLa cells were both reverse transfected during plating and transfected after pre-plating the cells with the siRNA targeting GAPDH or negative control siRNA. 48 hours post-transfection, GAPDH expression was measured by real-time RT-PCR. Percent gene expression was calculated as GAPDH gene expression in GAPDH siRNA transfected cells compared to those transfected with the negative control siRNA.
As shown in Figure 1, the reverse transfection method provided significant reduction in GAPDH mRNA in all five cell types with as little as 1 nM siRNA. Interestingly, reverse transfection required approximately three-fold less siRNA to induce the same reduction in GAPDH mRNA expression as the corresponding cells transfected using the standard procedure (Figure 1). In addition, while standard pre-plated transfection was limited to transfecting 510 x 103 cells per well on a 96 well plate, reverse transfection exhibited similar reductions in target mRNA expression using 525 x 103 cells (data not shown).
Reproducibility of Reverse Transfection. To address reproducibility, we reverse transfected cells in eight wells of each of three different 96 well plates with 10 nM GAPDH siRNA. Two days later, we repeated the procedure with three more 96 well plates. Analysis of GAPDH mRNA 48 hours after transfection revealed that the reverse transfection procedure was indeed highly reproducible (Figure 2).
Figure 2. Reproducibility of Reverse Transfection Procedure. Silencer GAPDH (Ambion) and Negative Control siRNA #1 (Ambion) (10 nM) were reverse transfected in triplicate (Plates 13; 8 wells/plate) into HeLa cells in a 96 well format. 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both target mRNA and 18S rRNA levels (Day 1). The entire experiment was repeated three days later (Day 3). Remaining gene expression was calculated as a percentage of target mRNA in cells transfected with siRNA targeting GAPDH compared to cells transfected with the negative control s iRNA. Data were normalized against the 18S rRNA signal. Data are presented as means SD.
Cell Viability Associated with Reverse Transfection. Cell viability for multiple cell types was measured under a broad range of conditions, including different transfection agent concentrations and siRNA concentration. Figure 3 shows cell viability post reverse transfection for a variety of siPORT NeoFX Transfection Agent amounts. In this cell type, siPORT NeoFX showed minimal toxicity. As transfection agent volume increased, greater levels of siRNA-mediated reduction of target gene expression were observed. Some transfection agents are not as flexible as siPORT NeoFX, and require more precision.
Figure 3. Transfection Agent Cytotoxicity. The indicated volumes of siPORT NeoFX Transfection Agent (Ambion) were used in reverse transfection of HepG2 cells. Assays were done in 96 well plates with 5 nM GAPDH siRNA (Silencer GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion). Remaining gene expression was calculated as a percentage of GAPDH mRNA in cells transfected with GAPDH siRNA compared to cells transfected with Negative Control siRNA. Data were normalized against the 18S rRNA signal. Cell viability (line) was measured using the ViaCount Assay (Guava Technologies).
A slight reduction in cell viability was observed in reverse transfected cells at higher siRNA concentrations. There was no distinguishable difference in cell health with <30 nM siRNA (data not shown). We suspect that the increased cytotoxicity at higher siRNA concentrations relates to the increased transfection efficiency of the method; we observe the same level of cytotoxicity when using standard transfection to deliver more than 100 nM siRNA.
96 Well Electroporation--For Primary Cells
While advances have been made in electroporation equipment and protocols , electroporation largely remains a process for single sample RNA and DNA delivery. We have created a device, the siPORTer-96 Electroporation Chamber, siPORT siRNA Electroporation Buffer, and associated protocols that enable the delivery of siRNAs to as many as 96 samples at once.
Efficiency and Cell Viability Associated with High Throughput Electroporation. We used the siPORTer-96 and siPORT siRNA Electroporation Buffer to deliver control siRNAs to several different cell types. The procedure consistently provided >70% reduction in target mRNA expression and >70% cell viability (Figure 4B). For many cell types, siRNA delivery efficiency and cell viability exceeded 90%. Time course studies using electroporation followed by real-time PCR revealed that target mRNA levels were reduced as soon as six hours after electroporation with maximal reduction at 2448 hours, depending on cell type (data not shown). Protein reduction lagged behind mRNA reduction with maximal protein reduction at 4872 hours post-transfection. Note that optimal electroporatio n conditionsnamely electrical pulse length, voltage, and number of pulsesare different for different cell types (see Figure 4A and our siPORTer Conditions Chart).
Figure 4. Gene Silencing and Cell Viability After Electroporation with the siPORTer-96 Electroporation Chamber. Silencer GAPDH (Ambion) or Negative Control #1 siRNA (Ambion) (1.0 g) were electroporated using the siPORTer-96 (Ambion) and siPORT siRNA Electroporation Buffer (Ambion) and conditions listed in panel A into 8 cell types. Conditions listed were for electroporating eight identical samples at a time. (panel B): 24 h later, the cells were harvested and analyzed by real time RT-PCR for GAPDH mRNA levels. 18S rRNA levels were used to normalize GAPDH expression. Remaining gene expression was calculated as a percentage of GAPDH mRNA levels relative to levels obtained from cells electroporated with negative control siRNA.
Reproducibility of High Throughput Electroporation. To assess well-to-well reproducibility of the high throughput electroporation system, a GAPDH siRNA (48 samples) or negative control siRNA (48 samples) were simultaneously electroporated into NHDF-neo cells using the siPORTer-96 and siPORT siRNA Electroporation Buffer. Reduction in GAPDH levels was monitored 48 hours later by real-time PCR. As shown in Figure 5, GAPDH mRNA was reduced by 9296% (average reduction: 94.8%). In addition, cell viability remained above 85% across the plate. These results indicated that the siPORTer-96 provided highly reproducible results. Unlike other electroporation chambers which often require a minimum of 1 x 105 cells to achieve acceptable delivery and enough viable cells for analysis, the combination of the siPORTer-96 and the siPORT siRNA Electroporation Buffer facilitated experiments with as few as 2.5 x 104 cells.
Figure 5. Reduction of GAPDH Gene Expression in Normal Human Dermal Fibroblasts-Neonatal after Electroporation using siPORTer-96 Electroporation Chamber. siRNA targeting GAPDH or a non-targeting siRNA (1.0 g) were electroporated into Normal Human Dermal FibroblastsNeonatal (an adherent primary cell type) using the siPORTer-96 Electroporation Chamber (Ambion) powered by a Bio-Rad Gene Pulser Xcell pulse generator. Samples 148 (shown) were transfected with Silencer GAPDH siRNA (Ambion) while samples 4996 were transfected with Silencer Negative Control #1 (Ambion) (data not shown). 48 h post-transfection, cells were harvested and target mRNA levels were analyzed by real-time RT-PCR. 18S rRNA levels were used to normalize GAPDH expression. Remaining Gene Expression (%) was calculated as a percentage of gene expression compared with the averaged value of cells transfected with the negative control siRNA.
Many siRNA applications require delivery of hundreds to thousands of siRNAs to cells with high efficiency while minimizing toxicity. Chemical reverse transfection with siPORT NeoFX Transfection Agent efficiently and reproducibly delivered siRNAs to immortalized, adherent cells in 96 and 384 well plates. In addition the siPORTer-96 Electroporation Chamber facilitated high throughput delivery of siRNAs to primary and immortalized suspension cells. While transfection efficiency and cell viability are cell type specific for both high throughput delivery methods, both methods were highly reproducible. Coefficients of variance, measured from the percent of mRNA remaining after siRNA delivery, were similar between 96 well reverse transfection and 96 well electroporation.
The combination of high throughput chemical reverse transfection using high performance reagents such as siPORT NeoFX and electroporation using the siPORTer-96 Electroporation Chamber, siPORT siRNA Electroporation Buffer, and optimized electroporation parameters, enables researchers to screen siRNA libraries to identify genes in virtually any cellular process using virtually any cell type. The widespread application of this approach will make it possible to assign cellular functions to many or even most of the genes in the human, mouse, and rat genomes.
Figure 6. Schematic Diagrams of Standard and Reverse Transfection Procedures.
Dmitriy Ovcharenko, Richard Jarvis, Scott Hunicke-Smith, Kevin Kelnar, and David Brown Ambion, Inc.