1. Preparing and counting the Namalwa cells
<ol>
	<li>Remove cells from culture flask and centrifuge the cells to pellet. Remove supernatant and resuspend cells in a known volume of PBS. The cells used in this protocol are suspension cells. If you are using adherent cells you will need to trypsinize before collecting.</li>
	<li>To determine the number of live cells, stain an aliquot of the cell suspension by mixing it 1:1 with a 0.4% trypan blue solution and pipet 10 &#956;l of the mixture onto a counting slide, and then insert the slide into the TC10 automated cell counter (Bio-Rad Laboratories, Inc).</li>
	<li>The TC10 cell counter will autofocus to assure the most accurate count, and then proceed with counting. It automatically determines the density of live cells and total cells in the sample.</li>
	<li>Select view image to display the cells on the viewscreen. Then select the dilution calculator to have the TC10 cell counter automatically calculate the amount of cell mixture that will be needed.</li>
	<li>This experiment is using a total of three siRNAs: one control siRNA and two different siRNAs targeting the STAT6 gene, plus untransfected controls. This requires 3.6 ml at a final concentration of 5 X 106 cells per ml.</li>
	<li>Enter the desired concentration and final volume values into the dilution calculator, and it calculates the volume of cell suspension needed based on the live cell count for that sample. Alternatively, perform the calculation manually. Then transfer the required volume of cell mixture for these experiments into a new tube.</li>
	<li>Spin the cell mixtures to pellet the cells and remove all the PBS. Resuspend in 3.6 ml of Gene Pulser electroporation buffer (Bio-Rad Laboratories). Transfer 800 &#956;l aliquots into tubes containing the appropriate siRNAs and mix gently. The cells are now ready for electroporation.</li>
</ol>
2. Electroporating the cells in 96-well Plates
<ol>
	<li>The 96-well electroporation plate allows the replicates for all four treatments to be electroporated together.</li>
	<li>Transfer 150 &#956;l of the cell suspension into the appropriate wells of a 96-well electroporation plate.</li>
	<li>Insert the plate in the MXcell electroporation plate chamber and close the lid.</li>
	<li>Electroporate using settings optimized for the cell line and buffer being used. These cells are electroporated using an exponential decay wave with instrument settings of 250 V, 350 &#956;F, and 1000 &#937; resistance.</li>
	<li>After electroporation is complete, pipette up and down gently in each well to mix and then transfer cells to a tissue culture plate containing pre-warmed media. Namalwa cells are grown in RPMI (Life Technologies) supplemented with 7.5% FCS and 1% pyruvate.</li>
	<li>Control samples that have not been electroporated are taken from the remaining cell suspension and added to culture dishes of pre-warmed culture medium identically to the electroporated cells.</li>
	<li>Incubate cells overnight at 37&#176;C with 5% CO2.</li>
	<li>After 24 hr, one set of cells was induced with 10 ng/ml final concentration recombinant IL-4 (R&#38;D Systems). The second set of control cells only receive additional media. All cells are incubated an additional 24 hr at 37&#176;C and 5% CO2.</li>
</ol>
3. Extract and check RNA
<ol>
	<li>After a total of 48 hr of growth, count the cells in each well, and transfer one aliquot from each well to a microfuge tube for RNA extraction, and freeze a second aliquot for western blotting or other protein analysis.</li>
	<li>Centrifuge samples to pellet the cells, and then remove and discard the supernatant from each tube.</li>
	<li>Proceed with the RNA extraction. We typically use the Aurum total RNA kit (Bio-Rad) according to the included protocol.</li>
	<li>When monitoring siRNA mediated gene knockdown it is particularly important to verify RNA quality because degraded RNA will give misleading results. The 260/280 ratio does not indicate RNA integrity therefore it is important to run the samples on a gel or microfluidic device to confirm RNA are not degraded.</li>
	<li>To assess both quality and quantity in one step, these samples will be analyzed using the Experion automated electrophoresis system (Bio-Rad).</li>
	<li>First, heat samples. Then, prime, load, and vortex the Experion RNA chip. Insert the chip into the Experion electrophoresis station and start run.</li>
	<li>After the run is complete, the Experion software will display results automatically in the form of an electropherogram, results table (that displays RNA concentration, ribosomal ratio, and the RNA quality indicator number), and virtual gel (which looks similar to a traditional RNA gel). High quality total RNA samples will typically have an RQI of 8-10.</li>
</ol>
4. Real-time PCR
<ol>
	<li>After verifying the RNA quality, proceed with the cDNA reactions. This experiment used the iScript one-step RT-PCR mix (Bio-Rad) to combine cDNA synthesis and real-time PCR in one reaction.</li>
	<li>Make a master mix containing all the reaction components other than the RNA templates for each primer set, and distribute those into the PCR plate.</li>
	<li>After diluting the RNA to a uniform concentration, add the RNA samples to the appropriate wells. Each reaction is set up in triplicate.</li>
	<li>Seal the plate, and place it into the CFX384 real-time PCR system (Bio-Rad). Select the appropriate protocol and begin the run.</li>
	<li>Once the run is complete, gene expression is determined by comparing gene expression of the gene of interest normalized to a reference gene in cells electroporated with experimental siRNA to the same normalization of cells electroporated with a control siRNA.</li>
</ol>
5. Analysis of siRNA knockdown
<ol>
	<li>The expression of CCL17 was monitored using quantitative real-time PCR to investigate the role of the transcriptional activator STAT6 in IL-4 dependent induction of CCL17. For this experiment, GAPDH was used as a reference gene. Alternate reference genes or multiple reference genes can be used in the same manner.</li>
	<li>The CFX Manager software automatically normalized CCL17 expression to expression of the reference gene from the same sample and automatically compared the resulting normalized values to control treatments by simply selecting GAPDH as &#34;reference&#34; and the control treatment (control siRNA without IL-4 induction) as &#34;control&#34; in the experiment settings window of the CFX manager software.</li>
	<li>Without IL-4 induction, transcription of CCL17 remained at low, constitutive levels comparable to the control treatment.</li>
	<li>Cells induced with IL-4 and retaining a normal STAT6 pathway (i.e., control siRNA or untransfected control treatments) showed 8-10 fold induction of CCL17 expression.</li>
	<li>Cells induced with IL-4 but with STAT6 expression inhibited by introduction of either STAT6 siRNA showed only about 2 fold higher CCL17 expression than the uninduced cells confirming the role of STAT6 in the IL-4 induced expression of CCL17.</li>
</ol>
6. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/1904/1904fig1.jpg" /> 
Figure 1. Overview of the IL-4 signaling pathway. 
A) Binding of IL-4 to its receptor leads to dimerization and activation of STAT6. Activated STAT6 translocates to the nucleus and activates transcription of its target genes, such as CC17. 
B) Without IL-4 induction, transcription of CCL17 will remain at low, constitutive levels. 
C) After siRNA-mediated silencing of the STAT6 gene, treatment with IL4 should lead to little or no increase in CCL17 expression.
<img alt="Figure 2" src="/files/ftp_upload/1904/1904fig2.jpg" /> 
Figure 2. Induction of CCL17 by IL4 measured by different conditions by quantitative real time PCR. 
A) Samples grown without IL-4 had a low constitutive level of expression regardless of gene expression. 
B) As shown in green and blue, control samples responded normally to treatment with IL-4; with an 8-10 fold induction of CCL17. However, cells transfected with siRNAs targeting STAT6, shown in red and pink, had diminished CCL17 expression in response to IL-4, supporting the idea that STAT6 plays a role in the IL-4 induced expression of CCL17.

The author is employed by Bio-Rad Laboratories that produces reagents and instruments used in this article.

This article has demonstrated how to electroporate siRNAs into a difficult to transfect suspension cell line and how to follow gene expression in these cells. When doing this procedure it s important to remember to keep things as consistent as possible across all the samples.

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.

Watch this Scientific Journal Video about Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells at JoVE.com

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

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 ...

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

Bacterial Gene Expression Analysis Using Microarrays

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...