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siPORT™ siRNA Electroporation Kit
Deliver siRNAs Into Primary Cells
• Deliver siRNA into primary cells, cells grown in suspension, and other difficult-to-transfect cells
• 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 [1]. 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 [2].
Now, Ambion introduces the siPORT
siRNA Electroporation Kit, which provides a Cy™3-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.
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| Figure 1. Electroporation
of Primary Cells and Hard-to-transfect Neuronal Cells. A
Cy™3-labeled GAPDH siRNA (1.5 µg)
was added to primary Human Mesenchymal Stem Cells (hMSC)
or rat neuronal pheochromocytoma (PC-12) cells in siPORT™ Electroporation
Buffer (75 µl) 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.
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Figure 2. siRNAs Electroporated into hMSC and NHDF-neo Cells. Six siRNAs (1.5 µg) targeting CDK2, JAK1, p53, GAPDH, uPAR, PKR, as well as a negative control siRNA, were electroporated into hMSC cells (A). Seven siRNAs (1.5 µg) 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).
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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 µg) 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.
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Summary
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.
Scientific Contributors
Dmitriy Ovcharenko, Po-Tsan Ku, Kevin Kelnar, Nitin Puri • Ambion, Inc.
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