Screening siRNA Libraries is Now More Feasible
While many laboratories are performing RNA interference (RNAi) screens with siRNA libraries to determine human gene function, the technology is not being applied as broadly as would be expected given its enormous potential. Three factors limit the adoption of this technology: (1) the cost of siRNA libraries, (2) the perception that expensive robotic liquid handling systems are required, and (3) the anticipated difficulties in performing transfections reproducibly in 96 well plates.
Ambion has addressed each of these issues by developing Silencer CellReady siRNA Libraries, siPORT NeoFX siRNA Transfection Agent, and the Silencer CellReady siRNA Transfection Optimization Kit (Figure 1). With this exciting new product line researchers can now perform RNAi screens for a fraction of the cost one might expect, meaning that almost d to make Ambions Silencer CellReady siRNA Libraries. This ensures that the siRNAs are stable and easy to resuspend.
Figure 3. Optimizing Transfection with the Silencer CellReady siRNA Transfection Optimization Kit. (A) Experimental design of a transfection optimization experiment using the Silencer CellReady siRNA Transfection Optimization Kit. (B) Knockdown of GAPDH levels were determined after the experimenets were performed as described in (A). In only two days, conditions that effectively delivered siRNAs into two cell types (HepG2 and A549) were identified.
To use, simply add the desired amount of transfection agent (0.4 ml siPORT NeoFX siRNA Transfection Agent is supplied, but other transfection agents can also be used), briefly incubate, and then overlay with the desired number of cells. Since the positive control siRNA targets GAPDH, a well-studied housekeeping gene expressed in all human cell types and often used as a control, target mRNA levels can be readily monitored using assays that most researchers already have available in their laboratories. siRNA induced knockdown of GAPDH protein can also be very quickly and easily monitored by the new KDalert GAPDH Assay Kit, which assesses GAPDH enzymatic activity via a fluorescence assay. (This kit, which was developed specifically for use with the Silencer CellReady siRNA Transfection Optimization Kit and Silencer GAPDH siRNA Controls, saves considerable time and money compared to standard real-time PCR protocols; for more information, see KDalert GAPDH Assay Kit: A New Way to Assess siRNA Delivery Efficiency). The convenient arrangement of siRNAs on each plate (Figure 3) can be used to assess the effectiveness of up to 24 different transfection conditions in duplicate. The Instruction Manual includes a plate map and step by step procedures for setting up optimization experiments.
Mike Byrom, Rich Jarvis, David Brown Ambion, Inc.
Cat# Product Name Size 1639 KDalert GAPDH Assay Kit 375 rxns 4510 siPORT NeoFX Transfection Agent 0.4 ml 4511 siPORT NeoFX Transfection Agent 1 ml 82xxx Silencer CellReady si RNA Libraries Varies 86050 Silencer CellReady siRNA Transfection Optimization Kit 3 plates + 0.4 ml transfection agent any lab can take advantage of RNAi screening.
Figure 1. Overview of RNAi Experiments Using the Silencer CellReady siRNA Library System. Step 1A: Search the Ambion website and choose a Silencer CellReady siRNA Library. Step 1B: Optimize siRNA transfection conditions for your cell line using the Silencer CellReady siRNA Transfection Optimization Kit. Step 2: Perform reverse transfection experiments in triplicate using the Silencer CellReady siRNA Library from Step 1A and the optimized conditions determined in Step 1B. Step 3: Assay cellular phenotype (e.g., cellular proliferation or apoptosis). Compare results from samples transfected with each gene-specific siRNA to those transfected with negative control siRNA. Step 4: Validate hits detected in Step 3 by a secondary assay.
Data Above: Gene Expression Results After Apoptosis and Cell Proliferation Screening of Silencer CellReady siRNA Plates Specific for CMGC Kinases. The data in Step 3 are from an actual screening experiment where CellReady sets of siRNAs, representing three individual siRNAs per target and targeting the CMGC subset of human kinases, were used to transfect HeLa Cells in triplicate using siPORT NeoFX. A confirmed hit was defined as any target gene that had two or more single siRNAs that induced a change in signal by > two standard deviations in the same direction as compared to cells transfected with Silencer Negative Control #1 siRNA. Signals were normalized by cell number. (A) Cells were assayed with alamarBlue (Biosource) 72 hr post transfection to identify kinases, that when silenced, affected cell pro liferation (orange bars). (B) In a separate experiment, cells were exposed to 25 M etoposide (Sigma) 24 hr post transfection. At 72 hr post transfection, levels of cellular apoptosis were monitored by assaying for caspase 3 activity.
Silencer CellReady siRNA Libraries
ready for transfection
EffectivesiRNAs designed for maximum potency and specificity
Economicala fraction of the cost of other siRNA libraries
For less money than you might think, the Silencer CellReady siRNA Libraries provide pre-defined sets of siRNAs in a ready-to-transfect format. Currently, sets targeting human kinases, the druggable genome, and two key gene sets are available, with Silencer CellReady siRNA Libraries for many additional targets available soon. Libraries are provided in sets of triplicate, ready-to-use plates, as well as in sets of 12 plates, permitting one and four screens, respectively, using triplicate transfections.
To produce the Silencer CellReady siRNA Libraries, Ambion uses the same high quality, high efficacy siRNAs from our standard Silencer siRNA Libraries and aliquots them into ready-to-use 96 well tissue culture plates. Using a proprietary procedure that ensures siRNA stability and ease of resuspension, the siRNAs are pre-plated and dried in the ideal quantity for use in a single RNAi screen in human cells.
Individual siRNAs for Enhanced Data Reliability
Three unique siRNAs are provided per target. Screening with three individual siRNAs per gene significantly decreases both false positive and false negative rates as compared to screening with pools of siRNAs, resulting in enhanced confidence in the data, reduced chances that important genes will be missed, and less time spent following up on false positive hits from the screen.
RNAi Screening Made Simple
To perform a screen with a Silencer CellReady siRNA Library, simply add diluted transfection agent to the dried siRNAs, incubate briefly (~10 minutes; the exact time depends on the transfection agent used), and overlay with cells. This process, in which cells are transfected and plated simultaneously, is referred to as reverse transfection. It eliminates the 24 hour wait after plating cells prior to standard transfection and has been found to be a more effective delivery method than standard transfection for many cell types (see the section on siPORT NeoFX below, and Optimizing siRNA Transfection for RNAi). Note: CellReady plates can be easily adapted for standard transfection as wel l.
Figure 1 (step 3) shows results from an RNAi screen in HeLa cells using a kinase-specific Silencer CellReady siRNA set. In this screen, several genes involved in cell proliferation and apoptosis were identified. A hit in this screen was defined as a gene whose inhibition by at least two of the three siRNAs tested resulted in a cellular phenotype that was significantly different (>2 standard deviations) than cells transfected with negative control siRNA. This simple experiment highlights the power of RNAi screening for determining gene function.
Silencer CellReady siRNA Library Format
Each Silencer CellReady siRNA Library includes sets of siRNAs targeting a particular human gene class or the druggable genome aliquotted into 96 well tissue culture plates. Three individual siRNAs per target are provided; the individual siRNAs to the same target are provided in separate plates in the same well location. The siRNAs are provided dried at 3 pmol per well, giving a final concentration of 30 nM when used under standard conditions. Replicates are provided for either 3 or 12 transfections. When ordering most sets, you can select four different control siRNAs from Ambions menu of popular positive and negative control siRNAs. These controls are provided in duplicate on each plate, allowing you to fully control your RNAi screening experiments. The final column of each plate remains empty for assay-specific positive and negative control siRNAs, as well as for non-treated control wells.
The first Silencer Screen-Ready siRNA Library sets include siRNAs for human kinases and for the druggable human genome. In addition, two popular gene setsone targeting a select group of kinases, and one targeting a select group of popular genesare available. Additional sets will be available soon. [View available Silencer CellReady siRNA Libraries]
siPORT NeoFX siRNA Transfection Agent
Versatileworks with a broad range of cell lines
Reproducibleproduces consistent results, lot to lot, plate to plate, and well to well
Fastreverse-transfects cells as they are plated, saving a day
Ideal for use with Silencer CellReady siRNA Libraries
The Ideal Reagent for RNAi Screens
siPORT NeoFX efficiently transfects adherent cells as they are sub-cultured, without increased cytotoxicity. It is the ideal companion for the Silencer CellReady siRNA Libraries because diluted siPORT NeoFX can be added directly to the wells of your library plates using a reverse transfection protocol. With this streamlined protocol and a Silencer CellReady siRNA Library, one person can perform >1000 individual transfections per hour without robotics.
siPORT NeoFX is compatible with most immortalized adherent cell types and is used routinely at Ambion to deliver siRNA into MCF-7 (human breast adenocarcinoma), HeLa-S3 (human cervix adenocarcinoma), HeLa (human cervix adenocarcinoma), HepG2 (human hepatocellular carcinoma), BJ (human foreskin fibroblast), A549 (human lung carcinoma), UMR106 (rat osteosarcoma), SKOv3 (human ovarian carcinoma), and SKNAS (human bone marrow neuroblastoma) cells.
Plate-to-Plate, Well-to-Well, and Lot-to-Lot Consistency in Performance
A good transfection agent is expected to reliably deliver nucleic acids at high transfection efficiencies across multiple wells for replicate samples, and among different lots of the transfection agent. Such characteristics are especially important in high throughput screening of siRNA libraries arrayed in 96 well plates. siPORT NeoFX shows negligible variability in gene silencing efficiency between multiple wells and multiple plates transfected with a positive control siRNA (Figure 2). This consistency is also evident in experiments performed on different days, and with different lots of reagent.
Figure 2. Consistent Day-to-Day, Plate-to-Plate, and Well-to-Well Performance of siPORT NeoFX. 8 x 103 HeLa cells were transfected in 96 well plates using siPORT NeoFX and either GAPDH siRNA (10 nM) or a negative control siRNA. Remaining GAPDH expression was quantified by r eal-time PCR. Each bar represents the mean of 8 replicate wells.
Two Convenient Sizes
siPORT NeoFX Transfection Agent is available in sizes of 0.4 ml and 1.0 ml, which is enough to perform up to 800 and 2000 transfections, respectively, in 96 well tissue culture plates. siPORT NeoFX is also provided as part of the Silencer CellReady siRNA Transfection Optimization Kit described below.
siRNA Transfection Optimization Kit
Perfect companion to Silencer CellReady siRNA Libraries
Demystifies and accelerates transfection optimization procedures
Includes three 96 well plates of pre-aliquotted GAPDH and negtive control siRNAs and 0.4 ml siPORT NeoFX Transfection Agent
Convenient and fastsimply add included transfection agent and cells, incubate, and assay
Efficiency of GAPDH siRNA delivery readily monitored by real-time PCR
Prior to siRNA library screening, optimal siRNA transfection parameters must be identified and verified for each cell type used. The Silencer CellReady siRNA Transfection Optimization Kit, which was developed specifically for optimizing transfection conditions in 96 well plates, dram atically simplifies this process.
Step by Step Instructions for Developing Optimal siRNA Delivery Conditions
Determining the ideal siRNA transfection parameters for a particular cell type can be a daunting task. Variables such as reagent identity, reagent volume, cell number, need for and timing of post-transfection wash, as well as timing of assay after transfection all need to be tested and controlled. The Silencer CellReady siRNA Transfection Optimization Kit provides pre-plated siRNA controls, siPORT NeoFX Transfection Agent, and a detailed Instruction Manual that leads you step by step through this process, reducing the time needed to complete transfection optimization.
Since the reagents are provided in a ready-to-use format, and the procedure is straightforward and described in detail, you will not be tempted to skip the optimization of critical parameters. The result: final transfection conditions that improve siRNA delivery.
Simple to Use, Easy to Assay Controls
At the heart of the Silencer CellReady siRNA
Transfection Optimization Kit are three 96 well plates containing multiple
aliquots of a validated positive control siRNA that targets human, mouse,
and rat GAPDH, as well as Silencer Negative Control #1 siRNA,
which is specifically designed to be a non-targeting, negative control.
These two siRNAs, each aliquotted into 48 wells of the 96 well plates
(Figure 3A), are pre-plated and dried using the same process use