Maintain high cell viability under a wide range of conditions
Optimize electroporation of your cells quickly with included positive and negative controls
Primary cells serve as an important model system for gene function analysis, target validation, and drug discovery since they are more similar to their in vivo counterparts than are immortalized cells. However, delivery of siRNA into these cells has proven to be a challenge. Efficient transfer of siRNA into primary cells by chemical transfection (e.g. using lipid-based reagents) works with only a few cell types . Ambion's new siPORT siRNA Electroporation Buffer overcomes this problem. It can be used with commonly available electroporators to ensure highly efficient siRNA delivery into many different cell types, including primary cells, while maintaining high levels of cell viability .
Now, Ambion introduces the siPORT siRNA Electroporation Kit, which provides a Cy3-labeled siRNA Control, and positive (GAPDH) and negative (non-targeting) siRNA controls in addition to the siPORT siRNA Electroporation Buffer. The kit makes it easy to optimize electroporation conditions for any cell type. For instance, successful delivery of siRNA into cells by electroporation depends on several critical parameters: voltage, pulse length, number of pulses, and siRNA concentration. The Cy3-labeled control siRNA makes it simple to monitor the uptake of siRNA into the target cells by fluorescence microscopy. The siPORT siRNA Electroporation Kit provides the means to dramatically simplify and speed up the electroporation optimization process, because a well-characterized siRNA targeting GAPDH and a widely used negative control siRNA and detailed optimization instructions are also included.
A Complete System for Optimization of siRNA Delivery
As seen in Figure 1, electroporation was used to deliver Cy3-labeled siRNA into human primary mesenchymal stem cells (hMSC) and rat neuronal pheochromocytoma (PC-12) cells. Almost every cell (>90%) contained detectable amounts of Cy3-labeled siRNA 24 hours after electroporation. This is a useful preliminary experiment to quickly screen multiple transfection conditions when working with new cell lines. A Cy3-labeled negative control siRNA comes with the kit for this purpose.
Figure 1. Electroporation of Primary Cells and Hard-to-transfect Neuronal Cells. A Cy3-labeled GAPDH siRNA (1.5 g) was added to primary Human Mesenchymal Stem Cells (hMSC) or rat neuronal pheochromocytoma (PC-12) cells in siPORT Electroporation Buffer (75 l) and electroporated using hMSC-specific or PC-12-specific parameters. Cells were fixed 24 hours after electroporation, stained with DAPI (blue), and analyzed by fluorescence microscopy (Cy3 fluorescence; red).
Because knockdown of gene expression in some cell types may not correlate 100% with uptake of fluorescently labeled siRNA, the siPORT siRNA Electroporation Kit also includes a positive control siRNA that targets GAPDH. The unlabeled GAPDH siRNA control can be used in conjunction with the unlabeled negative control siRNA (also included) to refine electroporation conditions by monitoring knockdown of GAPDH mRNA levels. The negative control siRNA is important not only for optimization experiments, but also for any siRNA experiment to confirm the absence of nonspecific effects due to transfection.
The siPORT siRNA Electroporation Kit has been tested with two commonly used single-cuvette electro-pulse generators: Gene Pulser Xcell (Bio-Rad) and ECM 830 (BTX). Their performance was similar when the same electroporation parameters were used.
Effective Silencing in Primary Cells
Figure 2 shows gene silencing induced by several different siRNAs, all delivered into hMSC and NHDF-neo cells (both are primary cells: human mesenchymal stem cells, and normal human dermal fibroblasts-neonatal) by electroporation using siPORT siRNA Electroporation Buffer. A critical parameter in the success of any RNAi experiment is the potency and specificity of siRNA sequences. To ensure the success of these gene-silencing experiments we used Silencer Pre-designed siRNAs. These siRNAs are designed using an extensively tested algorithm developed by Cenix BioScience, and have been shown to be highly effective at silencing target genes. As seen in Figure 2, 48 hours after transfection, mRNA expression levels of the targeted genes were reduced by 70% or more for all siRNAs tested in both cell types.
Figure 2. siRNAs Electroporated into hMSC and NHDF-neo Cells. Six siRNAs (1.5 g) targeting CDK2, JAK1, p53, GAPDH, uPAR, PKR, as well as a negative control siRNA, were electroporated into hMSC cells (A). Seven siRNAs (1.5 g) targeting Cyclin D1, NFκBp65, STAT1, PKCα, JAK1, RAF1, MMP-2, and a negative control siRNA were individually electroporated into NHDF-neo cells (B). All electroporations were conducted using the siPORT siRNA Electroporation Kit. 48 hours after electroporation, the cells were harvested and analyzed by real-time RT-PCR for corresponding gene expression levels. 18S rRNA levels were used to normalize the expression data. Percent remaining gene expression was calculated as a percentage of target mRNA compared with target mRNA from cells electroporated with the negative control siRNA.
Effective Gene Silencing in Primary and Difficult-to-Transfect Cells
In Figure 3, several different cell types were electroporated with the GAPDH control siRNA using individually optimized electroporation conditions. 48 hours after electroporation, GAPDH mRNA levels were reduced by 70% or more in each cell type. Cell viability of 70% or greater was maintained (Figure 3). Ambion scientists have optimized electroporation conditions for many different cell types. These electroporation conditions are available on our Electroporation Buffer webpage (see uppermost data table on right side of page).
Figure 3. Cell Viability and Reduction of GAPDH Gene Expression in Several Cell Types. Successful gene silencing and high cell viability was achieved in 11 primary cell types and 3 hard-to-transfect immortalized cell lines: Primary Cells: human mesenchymal stem cells (hMSC), normal human astrocyte cells (NHA), normal human dermal fibroblasts-neonatal (NHDF-Neo), rat astrocytes (DI TNC1), normal human umbilical vein endothelial cells (HUVEC), bovine aortic endothelial cells (BAEC), bovine aortic vascular smooth muscle cells (BAVSMC), bovine adrenal microvascular endothelial cells (BAMEC), mouse embryo fibroblast (MEF), rhesus monkey stem cells (RMSC), bovine lung microvascular endothelial cells (BLMVEC) are shown in blue. Hard-to-transfect cell lines: Jurkat (human acute T-cells), K562 (human erythroleukemia cells), PC12 (rat pheochromocytoma cells) are shown in black.
siRNA targeting GAPDH or a negative control siRNA (1.5 g) were electroporated. 48 hours after transfection, the cells were harvested and analyzed by real time RT-PCR for gene expression levels. 18S rRNA levels were used to normalize GAPDH expression. Remaining gene expression was calculated as a percentage of gene expression compared with the negative control siRNA.
Ambion's siPORT siRNA Electroporation Kit provides a streamlined protocol, with the necessary reagents for delivering siRNAs into multiple cell types. Because some biologically important cell types are refractory to other methods of transfection, electroporation can greatly simplify siRNA studies in these cell lines.
Dmitriy Ovcharenko, Po-Tsan Ku, Kevin Kelnar, Nitin Puri Ambion, Inc.