Navigation Links
High Throughput siRNA Electroporation

NEW
siPORTer-96 Electroporation Chamber

Efficient siRNA delivery with minimal cell mortality
Electroporate up to 96 samples at the same time
Deliver siRNA into primary, hard-to-transfect, and suspension cells
Does not require expensive, consumable electroplates or cuvettes
Use with Bio-Rad Gene Pulser Xcell pulse generator


Electroporation: An Advantageous Alternative to Chemical Transfection

Human primary cells are often preferred for RNAi experiments because they are more similar to their in vivo counterparts than are immortalized cells. Although common chemical transfection methods are effective in many transformed cell lines, they are often not effective for primary cells [1]. For these cell types as well as suspension and other hard-to-transfect cells, electroporation is a valuable alternative.

Electroporation involves applying an electric field pulse to induce the formation of microscopic pores in the cell membrane. These pores allow nucleic acids such as siRNA to traverse the membrane. Under specific pulse conditions, the pores reseal, and the electroporated cells recover and resume growth.

The siPORTer-96 Electroporation Chamber (patent pending; Figure 1) works with the Bio-Rad Gene Pulser Xcell pulse generator to allow high throughput siRNA electroporation. The specially designed electrodes enable rapid electroporation without requiring expensive, consumable electroplates or cuvettes. When you use the siPORTer-96 Electroporation Chamber to deliver siRNA, you will be able to detect RNAi-induced gene silencing just a few hours after delivery [2] (also, see Deliver siRNAs Into Primary Cells).

Figure 1. siPORTer-96 Electroporation Chamber. This photograph shows the electroporation chamber base with the electrode cartridge. The siPORTer-96 Electroporation Chamber was designed for safety and convenience. The assembled electrode cartridge protects samples from contamination--samples can be loaded inside a laminar flow hood, and the assembled electrode cartridge can be carried to the electroporation chamber base. Alternatively, the entire device can be set up inside a laminar flow hood.


Greater Cell Viability

Most existing electroporation protocols were developed to deliver plasmid DNA into cell nuclei. These protocols often result in high rates of cell mortality. Since siRNAs need only be delivered into the cytoplasm, milder electroporation conditions can be used. Using milder electroporation conditions and siPORT siRNA Electroporation Buffer (included) minimizes cellular mortality and trauma without sacrificing efficient siRNA delivery [2]. With electroporation, transfection occurs immediately, and cells can be transfected regardless of their cell cycle stage.


High Throughput, Reproducible Results

A multiwell electroporator makes optimizing electroporation conditions for new cell types much easier than single well electroporators. In addition, high throughput electroporation of siRNA libraries into primary cells is an extremely effective research tool for investigating gene function across entire gene classes. The siPORTer-96 Electroporation Chamber allows screening of large numbers of siRNAs rapidly, reproducibly, and in parallel. Using the same pulse conditions in a single run, other parameters can be identical or varied across wells. The standard sample volume is just under 50 l, and as few as 2.5 x 104 cells can be used per reaction. This means that ~100 siRNAs could be screened using only 2.5 x 106 cells and 4.4 ml of siPORT siRNA Electroporation Buffer.

The siPORTer-96 Electroporation Chamber yields results that are remarkably reproducible (Figure 2). 96 samples were electroporated using identical conditions. Half of the samples received an siRNA targeting GAPDH (Silencer GAPDH siRNA, Cat# 4605), and half received a scrambled Negative Control siRNA (Silencer Negative Control #1 siRNA, Cat# 4611). Data are shown for the GAPDH siRNA-treated samples. Normal expression levels of GAPDH were reduced by a mean of 94.8% with a coefficient of variance of 18.9%. Variability is attributed to steps in the procedure such as pipetting error, measurement error, and assay error, as well as true biological variability.

Figure 2. Reduction of GAPDH Gene Expression in NHDF-neo Cells. siRNA (1 g) was electroporated into NHDF-neo (normal numan dermal fibroblast cells, an adherent primary cell type) using the siPORTer-96 Electroporation Chamber. Samples 1-48 were transfected with an siRNA targeting GAPDH (Silencer GAPDH siRNA, this figure), and samples 49-96 were transfected with a negative control siRNA (Silencer Negative Control #1 siRNA, data not shown). 48 hours after transfection, the cells were harvested and analyzed by real-time RT-PCR for GAPDH expression levels. 18S rRNA levels were used to normalize GAPDH expression. Remaining Gene Expression was calculated as a percentage of GAPDH gene expression compared with the averaged value from cells transfected with the negative control siRNA.


Figure 3 shows results of experiments used to identify optimal electroporation conditions for NHDF-neo primary cells. An siRNA targeting GAPDH and a Negative Control siRNA were again used for this experiment. Both remaining GAPDH expression levels and cell viability were measured. These data illustrate the impressive gene silencing that can be achieved by delivering a potent siRNA with the siPORTer-96 Electroporation Chamber while maintaining cell viability. A wide range of parameters are effective in delivering siRNA to adherent cells like NHDF-neo cells.

Figure 3. GAPDH Gene Silencing and Cell Viability Under Various Electroporation Conditions. siRNA (1 g) targeting GAPDH (Silencer GAPDH siRNA, Ambion) or a Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion) were electroporated into normal human dermal fibroblast cells (an adherent primary cell type) using the siPORTer-96 Electroporation Chamber. Four samples were electroporated at a time, either with an siRNA targeting GAPDH (two samples) or with the negative control siRNA (two samples). The voltage was varied between 200 and 600 V for either 250 or 400 sec as labeled. 24 hours after electroporation, the cells were harvested and analyzed by real-time RT-PCR for GAPDH expression levels. 18S rRNA levels were used to normalize GAPDH expression. Remaining Gene Expression was calculated as a percentage of GAPDH gene expression compared with samples transfected with the negative control siRNA (bars). Cell viability was measured with ViaCount Cell Viability assay (Guava) (diamonds).


Summary

When used with Bio-Rad's Gene Pulser Xcell pulse generator, Ambion's siPORTer-96 Electroporation Chamber enables efficient high throughput electroporation of siRNA. Coupled with the siPORT siRNA Electroporation Buffer, the electroporator provides highly efficient siRNA delivery into primary and other hard-to-transfect cell types with minimal cell mortality. Standard sample volume is 44 l, and enough siPORT siRNA Electroporation Buffer (12 X 1.5 ml) is supplied with the siPORTer-96 Electroporation Chamber for 360 samples. Conditions can be identical or varied across samples, and multiple wells allow for faster condition optimization. There is no need for expensive, disposable electrode plates or cuvettes. The unit is simply wiped with ethanol to clean.


Scientific Contributors
Dmitriy Ovcharenko, Rich Jarvis, Po-Tsan Ku, Kevin Kelnar, Vince Pallotta, Scott Hunicke-Smith Ambion, Inc.


back to top


Ordering Information
Cat# Product Name Size 8990 siPORT siRNA Electroporation Buffer 12 x 1.5 ml 13500 siPORTer-96 Electroporation Chamber 1 device
'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. Innovative Tissue Array Technology for High-Throughput Screening of Gene Expression
2. Highest Possible Transformation Efficiencies for High-Throughput Applications
3. High-Throughput Cultivation of Bacterial Clones
4. High-Throughput Isolation of Total RNA
5. High-Throughput System Generates Ultra-Pure PCR Products Suitable for All Applications
6. The DIG System Nonradioactive Automated High-Throughput In Situ Hybridization: a Powerful Tool for Functional Genomics Research
7. High Throughput aRNA Amplification
8. High Throughput siRNA Delivery In Vitro: From Cell Lines to Primary Cells
9. High Throughput RNA Isolation for High Performance Microarray Analysis
10. RNAi for High Throughput Analysis
11. Robust High Throughput RNA Isolation from Blood Samples
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: High Throughput siRNA Electroporation

(Date:7/31/2014)... organs and even the entire body to visualize long-range ... has been a long-time dream of biologists. A study ... the journal Cell has now made that ... organs, bodies, and human tissue biopsies transparent, while keeping ... pave the way for a better understanding of brain-body ...
(Date:7/31/2014)... Indiana University Bloomington have described the self-assembly of ... with potential value for the field of organic ... cells. , Their paper, "Anion-Induced Dimerization of 5-fold ... Self-Assembled Crystals," has been published online by ... Society of Chemistry. It is the first collaboration ...
(Date:7/31/2014)... , July 31, 2014 ... Market Research "Quantum Dots Market - Global Industry Analysis, ... the market was valued at USD 88.5 million in ... by 2023, growing at a CAGR of 53.8% from ... Dots Market Report at  http://www.transparencymarketresearch.com/quantum-dots.html Increasing ...
(Date:7/31/2014)... July 31, 2014 Graphite Metallizing Corporation ... temperature oven carts. These bearings are uniquely suited to ... ball bearings because of the high temperatures . ... help from the design engineers at Graphite Metallizing to ... process. During their manufacturing operation, pans containing blocks of ...
Breaking Biology Technology:See-through organs and bodies will accelerate biomedical discoveries 2Chemists demonstrate 'bricks-and-mortar' assembly of new molecular structures 2Chemists demonstrate 'bricks-and-mortar' assembly of new molecular structures 3Global Quantum Dots Market is Expected to Reach USD 8,246.8 Million in 2023: Transparency Market Research 2Global Quantum Dots Market is Expected to Reach USD 8,246.8 Million in 2023: Transparency Market Research 3Global Quantum Dots Market is Expected to Reach USD 8,246.8 Million in 2023: Transparency Market Research 4Graphite Metallizing Announces Bearings for High Temperature Oven Applications 2Graphite Metallizing Announces Bearings for High Temperature Oven Applications 3
... Moralejo Garca at the University of the Basque Country (UPV/EHU), ... other magnetic devices of wide industrial application. , It is ... are ever smaller and that nanotechnology, although it its infancy, ... our society. Who has not heard of nanotubes, nanoparticles and ...
... Announces Milestone in Wall Street Presentation, INDIANAPOLIS, ... and Sangamo BioSciences, Inc. (Nasdaq: SGMO ) ... milestone as part of,their joint Research and Commercial ... Sangamo,s zinc finger DNA-binding,protein nuclease (ZFN(TM)) technology in ...
... CORD:USE Cord Blood Bank,is proud to announce that ... joined its team. "We,re honored to have this,NASCAR superstar ... President and CEO of CORD:USE. Jimmie Johnson joins CORD:USE ... Race for Life(TM)",campaign., "I,m excited about telling people ...
Cached Biology Technology:A researcher of UPV/EHU has designed nanomagnets for industry 2Dow AgroSciences, Sangamo BioSciences Announce the Achievement of Key Milestone in Plant Agriculture Collaboration 2Dow AgroSciences, Sangamo BioSciences Announce the Achievement of Key Milestone in Plant Agriculture Collaboration 3Dow AgroSciences, Sangamo BioSciences Announce the Achievement of Key Milestone in Plant Agriculture Collaboration 4Two-time Defending NASCAR Sprint Cup Series Champion Jimmie Johnson Joins the Team at CORD:USE Cord Blood Bank to Help Win the Race for Life 2
(Date:7/30/2014)... truffle uses reversible epigenetic processes to regulate its genes, ... - a picture of the genome regulation taking place ... journal Genome Biology and illustrates how the ... ,jumping genes,. The authors say this may shed light ... , Black truffles (Tuber melanosporum), also known as Prigord ...
(Date:7/30/2014)... effects of climate change on the world,s animal ... factors, according to a new paper by a ... of Queensland, and other organizations. The authors claim ... the point when it comes to climate change. ... scientists focus on the "direct" threats of changing ...
(Date:7/30/2014)... Antonio Life Sciences Institute (SALSI), a joint venture between ... The University of Texas Health Science Center at San ... Pilot Program. The winners, Emily Boice from UTSA and ... $25,000 for their project titled, "Novel engineered ferritins for ... , Boice and Huang,s research is focused on finding ...
Breaking Biology News(10 mins):How black truffles deal with the jumpers in their genome 2Conservation scientists asking wrong questions on climate change impacts on wildlife 2
... 30 years with Chip Supply, Inc., President & CEO ... Perrott served in various capacities through the years, becoming ... Supply grew into the largest independent die distributor in ... of high reliability interconnect solutions, serving the military, aerospace, ...
... fulfill a range of diversified functions. Often such specialization ... places within a cell. It ensures that particular functions ... intracellular asymmetry in the otherwise fluid environment of the ... represent an especially important type of freight. They are ...
... through nearly 390,000 chemical compounds had led scientists to ... surrounding the brain with effects promising for new drugs ... substance, which blocks formation of cholesterol in the brain, ... Aleksey G. Kazantsev and colleagues previously discovered that blocking ...
Cached Biology News:How molecules get to the right place at the right time 2How molecules get to the right place at the right time 3