Navigation Links
Deliver siRNAs Into Primary Cells

siPORT siRNA Electroporation Kit
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 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 afte r 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 main tained (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.


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.


back to top


Ordering Information
Cat# Product Name Size 1629 siPORT siRNA Electroporation Kit 60 rxns 8990 siPORT siRNA Electroporation Buffer 12 x 1.5 ml
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. pFB-ERV: Retroviral Delivery of the Ecdysone Receptor Proteins
2. High-Titer Retroviral Vectors for Gene Delivery
3. Unique Enhanced DNA Polymerase Delivers High Fidelity and Great PCR Performance
4. A New Way to Assess siRNA Delivery Efficiency
5. Delivering siRNA Using Adenoviral Vectors
6. Efficient Delivery of siRNAs to Human Primary Cells: Electroporation vs. Chemical Transfection
7. Delivering siRNAs to Difficult Cell Types
8. Enhanced siRNA Delivery and Long-term Gene Silencing
9. High Throughput siRNA Delivery In Vitro: From Cell Lines to Primary Cells
10. Delivery of pCMV-S DNA Using the Helios Gene Gun System Is Superior to Intramuscular Injection in Balb/c Mice
11. Silencer Validated siRNAs
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:3/23/2017)... ... ... is proud to announce it has become the premiere team-building cooking event company in San ... world, such as Illumina, HP and Qualcomm, and is ranked #1 in its category on ... to its new team building format, a way for teams to not only interact with ...
(Date:3/23/2017)... -- BioPharmX Corporation (NYSE MKT: BPMX), a specialty pharmaceutical ... reported financial results for the quarter and year ... update on the company,s clinical development efforts and ... pleased to report that last year was a ... Krammer. "We achieved key clinical milestones and attracted ...
(Date:3/23/2017)... , March 23, 2017  Agriculture technology company Cool ... financing and note conversion to commercialize its Cool Terra ... focused on developing products that are simultaneously profitable as ... in the last 18 months. This latest round of ... Venture Partners. The company,s primary product, ...
(Date:3/22/2017)... March 22, 2017  Ascendis Pharma A/S (Nasdaq: ... TransCon technology to address significant unmet medical needs ... the full year ended December 31, 2016. ... our company as we broadened our pipeline and ... rare disease company with an initial focus on ...
Breaking Biology Technology:
(Date:2/16/2017)... 2017  Genos, a community for personal genetic ... received Laboratory Accreditation from the College of American ... laboratories that meet stringent requirements around quality, accuracy ... "Genos is committed to maintaining the ... honored to be receiving CAP accreditation," said ...
(Date:2/13/2017)... Former 9/11 Commission border counsel and Special ... Kephart of Identity Strategy Partners, LLP, today releases ... "Executive Order: Protecting the Nation From Foreign Terrorist Entry ... "As President Trump,s ,Travel Ban, Executive Order gains more ... the travel ban, it is important that our national ...
(Date:2/8/2017)... Feb. 8, 2017 About Voice Recognition Biometrics ... match it against a stored voiceprint template. Acoustic ... cadence, and tone are compared to distinguish between ... installation, as most PCs already have a microphone ... Voice recognition biometrics are most likely to be ...
Breaking Biology News(10 mins):