1. MEA Preparation
<ol>
	<li>MEAs for use in electroporation experiments can either be fabricated using standard photolithography technique as described previously18 or purchased directly from vendors of MEA manufacturers such as Multi Channel Systems <a href="http://www.multichannelsystems.com" target="_blank">(http:/www.multichannelsystems.com/)</a> and Alpha MED Scientific, Inc. <a href="http://www.med64.com/" target="_blank">(http://www.MED64.com)</a>.The MEAs used in our experiments were fabricated in-house at the clean room facility in Arizona State University, which is administered by the Center for Solid State Electronics Research (CSSER). The MEAs used in our experiments had 32 indium tin oxide electrodes in an 8 by 4 array with electrode diameter sizes from 50-200 &mu;m within a 1 cm inner diameter glass well. The glass well was bonded on the MEA using polydimethylsiloxane (PDMS).</li>
	<li>Sterilize the MEA in an autoclave prior to cell seeding. Other methods of sterilization such as a soaking in 70% ethanol, UV treatment and hot water treatment as described elsewhere can also be used as long they do not compromise the integrity of the MEA.</li>
	<li>Transfer the autoclaved MEA to a laminar flow hood and place it in a standard sterile polystyrene Petri dish. Prior to seeding cells on the MEA, sterilize the laminar flow hood by turning on the UV light for 20 min. If the MEA is sensitive to UV light cover the Petri dish containing the MEA with sterile aluminum foil while the flow hood is under UV illumination.</li>
</ol>
2. Seeding Cells on the MEA
<ol>
	<li>Harvest cells from a running cell line of adherent mammalian cells using Trypsin/EDTA and raise them to a concentration of 100-200 cells/&mu;l in 1 ml cell media for plating. In our experiments we have been successful in culturing NIH 3T3 fibroblasts cell line, HeLa cell line and primary cortical and hippocampal neurons on the MEA.</li>
	<li>Place a 20-30 &mu;l drop of media with cells on the MEA. Transfer the Petri dish with the MEA to the incubator. It is important to ensure that the cell drop size is large enough to cover all the electrodes on the MEA.</li>
	<li>2 hr after seeding the cells transfer the Petri dish with the MEA from the incubator to laminar flow hood and gently add 400-500 &mu;l of pre warmed media (37 &deg;C) to the MEA well. Check under the microscope to ensure that cells are still attached and evenly spread on the MEA surface. Transfer the MEA back to the incubator. The 2 hr wait period before the addition of media ensures enough time for the cells to adhere with the MEA surface. Addition of cell media too soon after seeding the cells can wash away the cells. If cells do not adhere well on the MEA, its surface can be treated with cell attachment factors such aspolylysine, fibronectin, and laminin to improve cell adhesion. In our HeLa cell experiments we typically treat the MEA surface with fibronectin (10 &mu;g/ml) for 1-2 hr.</li>
	<li>8-12 hr after seeding the cells, perform 1-2 washes with PBS to remove any dead cells. Cell medium can replaced every 24-48 hr as needed. In a typical experiment, the cells are usually ready for transfection 24-48 hr after seeding. To maintain consistency in electroporation efficiency it is recommended to wait until the culture is at least 80% confluent before electroporation.</li>
</ol>
3. Site-specific Transfection of siRNA
<ol>
	<li>Prepare nucleic acid solution. For siRNA transfection, prepare a 1-2 &mu;M siRNA electroporation solution in ice cold electroporation buffer to attain a final volume of 200-400 &mu;l. In this video article we demonstrate the transfection of HeLa cells with Alexa 488 conjugated scrambled sequence of siRNA.</li>
	<li>Transfer the MEA with cells from the incubator to sterile laminar flow hood. Remove the cell media from the MEA and perform a wash with PBS. Thereafter, add 200-400 &mu;l of the ice cold 1 &mu;M siRNA electroporation solution to the well.</li>
	<li>Apply anodic square electric pulses to the targeted electrodes (Refer to step 4 for determining optimal parameters of the electric pulse). In our experiments we used a Pragmatic Instruments 2414A waveform generator to generate electric pulses and a dual-inline-package (DIP) 16 pin clip to make contact with the MEA bond pads. The waveform generator can be connected to the DIP clip via a demultiplexor printed circuit board to provide selectivity of individual electrodes. For the reference electrode, place a steel cathode in the cell media. While lowering the steel reference electrode in the media it is extremely important to ensure that it does not make contact with the MEA surface. Upon contact, it can cause damage to the cells and the MEA. It is recommended to lower the reference electrode to a position of 1 mm above the cells. A spacer with a height of 1 mm can be placed in the well to assist in placement of the reference electrode. An alternate approach to using an external reference electrode is to use neighboring electrodes as cathode-anode pairs.</li>
	<li>Immediately after applying the electric pulses to the targeted electrodes on the MEA, transfer the MEA back to the incubator. After an incubation period of 5 min, gently replace 75% of the electroporation buffer with pre-warmed (37 &deg;C) cell media. Later, fluorescent imaging can be done to confirm the delivery of fluorescently tagged siRNA to the cells.</li>
</ol>
4. Electroporation Parameter Optimization
<ol>
	<li>To determine the optimal electric pulse parameters for electroporation, design electroporation experiments for a range of values of pulse amplitude, pulse duration, and number of pulses. In our experience with HeLa cells, we observed that a single voltage pulse of amplitude from 3 V to 9 V, and duration from 1 msec to 10 msec for an electrode size of 100 &mu;m led to successful electroporation with minimal cell death. It is our recommendation that one parameter be varied at a time and others are kept constant. To quantitate the electroporation efficiency for each of the pulse parameters, we use propidium iodide (PI), a cell impermeant dye that stains nucleic material, and live assay based methodology, as described in steps 4.2-4.5.</li>
	<li>Establish a cell culture on the MEA following the directions outlined in steps 1 and 2.</li>
	<li>Prior to electroporation prepare a 300-500 &mu;l of 30 &mu;g/ml PI solution in ice cold electroporation buffer and 400 &mu;l of 4 &mu;M calcien AM solution in PBS.</li>
	<li>Transfer the MEA from the incubator to the laminar flow hood. Remove the cell media from the MEA and perform a wash with PBS. Thereafter, add 300-500 &mu;l of the ice cold electroporation buffer with PI.</li>
	<li>Apply electric pulses with desired pulse amplitude and duration to the targeted electrodes (The equipment used in our experiments for generating and applying the electric pulse to the MEA is described in step 3.3). For the reference electrode either lower a steel electrode into the electroporation buffer in the MEA well or use the neighboring electrode as reference. By selectively applying pulses of varying amplitudes or durations to different electrodes multiple experiments can be performed on the same device. For example, on a 32 electrode MEA, 8 experiments can be performed by splitting the 32 electrodes in 8 groups, each consisting of 4 electrodes. Each of the 8 groups can then be stimulated by a single rectangular voltage pulse of duration 5 msec with amplitudes varying in 3 V-6 V range in 0.5 V increments and one group can be used as a control.</li>
	<li>Immediately after applying the voltage pulses transfer the MEA to the incubator for 5 min. Afterwards, gently remove 75% of the electroporation buffer from the MEA well and replace it with warm cell media. Transfer the MEA back to the incubator.</li>
	<li>After an incubation period of 2-4 hr replace the cell media in the MEA well with 300-500 &mu;l of 4 &mu;M Calcein AM solution in PBS. Transfer the MEA back to incubator for another 30 min.</li>
	<li>Following the incubation of the cells with Calcein AM, replace the Calcein AM solution in the MEA well with PBS after two washes with PBS. Obtain fluorescent images of cells using with an optical microscope with filters for PI (Excitation/Emission - 535 nm/617 nm) and Calcein AM (Excitation/Emission - 490 nm/520 nm). The uptake of PI, due to electroporation, causes cells to fluoresce red and the Calcein AM causes cells that are alive to fluoresce green. Three possible scenarios that can be observed are 1) cell electroporated and alive (will fluoresce both red and green), 2) Cells alive but not electroporated (will fluoresce just green) and 3) Cells dead due to electroporation (will fluoresce just red). The cell viability was calculated as a percent of the total number of cells alive on the electrode and the electroporation efficiency was calculated as percent of the total number of cells loaded with PI/siRNA.</li>
</ol>
5. Representative Results
Figure 1 shows the site-specific loading of HeLA cells with PI. It can be observed that only cells on the electrode demonstrate an uptake of PI (Figure 1B). A live assay performed post electroporation was used to assess the viability of cells (Figure 1A). The electroporation efficiency and cell viability for the example shown in Figure 1 are 81.8 % and 96.1 % respectively. High cell viability and electroporation efficiency can be achieved by optimizing the electroporation pulse parameters. Site-specific transfection of HeLA cells with fluorescently tagged siRNA for an optimized electroporation pulse (8 V, 1 msec) is shown in Figure 2. The electroporation efficiency for the siRNA at 8 V, 1 msec was found to be 74.4 &plusmn; 16.0 % and cell viability was found to be 87.9 &plusmn; 8.4 % (all data represented as mean &plusmn; standard deviation, N=5).
<img alt="Figure 1" src="/files/ftp_upload/4415/4415fig1.jpg" /> 
Figure 1. Site-specific delivery of PI in HeLa cells on a 200 &mu;m electrode (6 V, 5 msec). A) Calcien based live assay performed 2 hr post electroporation. Alive cells are indicated by green fluorescence. B) Red fluorescence demonstrates the uptake of PI by cells on the electrode. C) A superimposed image of A and B. The white arrows indicate cells that are electroporated and alive. The black arrows indicate cells that are dead. The white circle in A, B and C indicates the outline of the electrode.
<img alt="Figure 2" src="/files/ftp_upload/4415/4415fig2.jpg" /> 
Figure 2. Transfection of HeLa cells with fluorescently tagged scramble sequence of siRNA. A) Image of HeLa cells on a 100 &mu;m electrode transfected with Alexa 488 tagged siRNA (8 V, 1 msec). B) Image of cell nuclei stained with DAPI. The white circle marks the edge of the electrode.

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No conflicts of interest declared.

In this video article we demonstrate the use of MEA for site-specific transfection of HeLa cells with scrambled sequence of siRNA. One of the advantages of this technology is its applicability to different cell lines including primary cell lines. Our lab has previously demonstrated the use of this technology for site-specific transfection of primary hippocampal neuronal culture from E18 day old rat and NIH-3T3 cells with scrambled siRNA sequences and GFP plasmid18, 19. We have also had success in delivering fu...

<table><tbody><tr><td>Cell media:&lt;br/&gt;Advanced MEM&lt;br/&gt;L-Glutamine 200 mM&lt;br/&gt;Penicillin/Streptomycin&lt;br/&gt;Fetal bovine serum</td><td>Gibco/Invitrogen&lt;br/&gt;Himedia Laboratories/VWR&lt;br/&gt;Lonza group Ltd.&lt;br/&gt;Gibco/Invitrogen</td><td>12492-013&lt;br/&gt;95057-448&lt;br/&gt;09-757F&lt;br/&gt;16000-044</td><td>Cell media composition:&lt;br/&gt; 2% FBS, 2%L-glutamine and 2% Pennstrep in Advance MEM</td></tr><tr><td>Trypsin EDTA</td><td>Mediatech, Inc.</td><td>25-053-CL</td><td></td></tr><tr><td>PBS</td><td>Mediatech, Inc.</td><td>21-040-CV</td><td></td></tr><tr><td>Alexa 488 and rhodamine tagged scrambled sequence siRNA</td><td>Qiagen, Inc.</td><td>1027292</td><td></td></tr><tr><td>Electroporation buffer</td><td>Biorad Laboratories</td><td>165-2677</td><td></td></tr><tr><td>Waveform generator</td><td>Pragmatic</td><td>2414A</td><td>Any waveform/pulse generator that can deliver the desired pulses can be used.</td></tr></tbody></table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

high-efficiency-site-specific-transfection-adherent-cells-with-sirna

Research

JoVE Journal

Bioengineering

13.4K Views.  Arizona State University. The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

Video: High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

Cancer Research

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Immunology and Infection

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Biology

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

TEEI Method for Electroporation Analysis and Delivery Prediction in A431 Cells

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

Author Spotlight: Optimizing Porous Substrate Electroporation Through Micro and Nanochannels for Enhanced Monitoring and Intermediate Stage Characterization

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3&#39;UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into &#62;75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.

We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

An Introduction to Transfection

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for transfected RNA is the cytoplasm, DNA must be transported to the nucleus for effective transfection. There, the DNA can be transiently expressed for a short period of time, or become incorporated into the genomic DNA, where the change is passed on from cell to cell as it divides.
This video describes the basics behind chemical mediated transfections and introduces some of the most commonly-used reagents, including charged lipids, polymers, and calcium phosphate. Each step is described from the preparation of cells for transfection through analysis of transfection efficiency. Additionally, the applications section of this video-article describes the use of electroporation and a biolistic transfection as alternative methods for introducing nucleic acid into mammalian cells. It also describes an advanced use of transfection where co-transfection of interfering RNA and DNA are introduced as a way to down-regulate a naturally occurring protein while at the same time producing a mutant variant of it within the same cell.

Transfection: Inserting Genetic Materials into Mammalian Cells

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell ...