Posttransfection Analysis of Cells

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en-us LUSOPS84 Posttransfection Analysis Posttransfection Analysis of Cells /webroot/web/html/lsr/solutions/technologies/transfection <p>This section describes different methods of analyzing cells after transfection, such as flow cytometry, fluorometry, laser-scanning molecular imaging, luminometry, real-time qPCR, microscopy, spectrometry, and western blot analysis. It also provides guidelines and methods that you can use for the analysis of transfected cells. The methods can be used to determine transfection efficiencies and to perform more in-depth analyses of the expression of your favorite gene/protein. However, depending on your cells, reagents, or equipment availability these methods should be modified to fit your laboratory needs.</p> <p><strong>Related Topics:</strong> <a href="/evportal/destination/solutions?catID=LUSONCB9O">Transfection,</a> <a href="/evportal/destination/solutions?catID=LUSONV30E">Instrument-Based Transfection Methods</a>, and <a href="/evportal/destination/solutions?catID=LUSOOP49">Chemical- and Viral-Based Transfection</a>.</p> General Considerations <p>There are many factors that can influence transfection efficiency, a number of which are specific to the target cell. Cell-related factors affecting transfection include cell density, cell size, replication state, passage number, health of cells, biomolecule type, and concentration. Some factors are method specific; for example, electroporation parameters such as voltage, capacitance, and resistance strongly affect transfection efficiency. Therefore, to obtain high efficiencies, all relevant factors should be considered when planning transfection experiments.</p> <p>Frequently, after any transfection experiment, it is important to assess the efficiency of transfection and the impact of transfection on the cells. Analysis of transfection efficiency can be as simple as confirming the expression of your gene of interest. However, in many cases assessment of transfection involves determining the total expression level of your gene of interest, determining the number of positive cells within a transfected population (% positive cells), and/or visual confirmation of your protein of interest (Jordan et al. 2007).</p> <p>Most methods for measuring protein expression level of your transfected cell will determine the total expression from a population of transfected cells. Measurement of total gene expression can be done through real-time quantitative PCR (real-time qPCR), western blot analysis, molecular imaging, and fluorometry. Determining the number of positive cells within a transfected cell population can be performed by microscopy and flow cytometry. Finally, confirming localization of your protein of interest can be done by microscopy.</p> <p>Reporter genes, such as green fluorescent protein (GFP), luciferase, or &beta;-galactosidase can be used to analyze transfection efficiency because their expression can be easily monitored. They can also be used to standardize transfection efficiencies between different transfection experiments by comparing the expression levels of their products. Reporter genes can be used alone or fused to a gene of interest to determine the protein expression level, the number of positive cells, or the location of the protein being studied.</p> <p>Following are some of the most common methods used for posttransfection analysis.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Flow Cytometry <p>A flow cytometer can determine the number of positive cells within a transfected cell population (% positive cells). In addition, a flow cytometer with sorting abilities can enrich for positive cell populations. However, flow cytometry requires that the cells either express a fluorescent protein, such as GFP, or your protein of interest must be labeled with a fluorescent molecule.</p> <p>For more information about flow cytometry, visit <a href="http://flowcyt.sourceforge.net/" target="_blank">http://flowcyt.sourceforge.net/</a>.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Expensive</li> <li>Can only detect fluorescence (often requires labeling protein with a fluorescent molecule)</li> <li>Time consuming</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Determines percentage of positive cells (quantitative measurement)</li> <li>Can enrich for positive cell population with sorting ability</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Fluorometry <p>A fluorometer can detect a wide range of fluorescence. Extracts from cells expressing fluorescent protein can be used to measure the expression level of your gene in the transfected cells.</p> <p>For more information about fluorescent protein detection in cell extracts, see Wilson J and Segal A (1998). Measuring intracellular enhanced green fluorescent protein with the VersaFluor&trade; fluorometer. Bio-Rad bulletin <a href="http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2368.pdf" target="_blank">2368</a>.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Can only detect a fluorescent molecule or protein</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <p>A fluorometer can detect a wide range of fluorescence. Extracts from cells expressing fluorescent protein can be used to measure the expression level of your gene in the transfected cells.</p> <p>For more information about fluorescent protein detection in cell extracts, see Wilson J and Segal A (1998). Measuring intracellular enhanced green fluorescent protein with the VersaFluor&trade; fluorometer. Bio-Rad bulletin <a href="http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2368.pdf" target="_blank">2368</a>.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Can only detect a fluorescent molecule or protein</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Laser-Scanning Molecular Imaging <p>Laser-scanning molecular imagers can detect a wide range of fluorescence. With the use of appropriate fluorophores, western blots or gels can be quantitatively analyzed with a scanner. In addition to analyzing western blots or gels, scanners can also directly detect and quantify lysate from a population of cells expressing a fluorescent protein, such as GFP. The ability to detect fluorescence allows the scanner to directly analyze cell lysate without running a gel.</p> <p>For more information about laser-scanning molecular imaging, see Bio-Rad bulletin <a href="http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2752.pdf" target="_blank">2752</a>. Applications for Molecular Imager FX Systems: Instrument settings.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Expensive</li> <li>Can only detect a fluorescent molecule or protein</li> <li>For quantitative analysis, need an internal control and densitometer</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Luminometry <p>A luminometer is an instrument that measures light intensity. Expression or co-expression of luciferase, which emits light after interacting with its substrate, can be used for quantitative analysis of total protein expression.</p> <p>For more information about luminometry and luciferase assays, see Sambrook J and Russell D (2001). Analysis of gene expression in cultured mammalian cells. In Molecular Cloning: A laboratory manual, Third Ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press), 17.96.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Luminometer needed</li> <li>Requires expression of luciferase gene (when luciferase assays are used)</li> <li>Only measures total gene expression from population of cells</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate with luminometer after addition of substrate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Microscopy <p>Microscopy allows direct visualization of transfected cells. Microscopy can be used to count the number of transfected cells for estimation of positive cell numbers. Microscopy can also be used to confirm the correct localization of your protein of interest. Visualization of cells transfected with your gene of interest can be accomplished in several ways. Expression or co-expression of a fluorescent protein, such as GFP, will allow the direct identification of your transfected cells. However, a non-fluorescent protein must be labeled for visualization. Immunofluorescence, chemical labeling, and immunohistochemistry techniques can be used to label proteins. In general, microscopy cannot be used for quantitative measurement of transfection efficiency unless you have image analysis software that can count and quantitate positively transfected cells.</p> <p>For more information about chemical and immunofluorescent labeling, fluorescence microscopy, and immunohistochemistry techniques, visit:</p> <ul> <li><a href="http://www.ihcworld.com/_protocols/immunofluorescence/immunofluorescence.htm" target="_blank">http://www.ihcworld.com/_protocols/immunofluorescence/immunofluorescence.htm</a></li> <li><a href="http://www.mwrn.com/microscopy/light/fluorescence.aspx" target="_blank">http://www.mwrn.com/microscopy/light/fluorescence.aspx</a></li> </ul> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Expensive</li> <li>Epifluorence microscope needed</li> <li>Must be able to visualize protein (often requires labeling protein)</li> <li>Time consuming if labeling protein is required</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Visual (direct observation of your protein of interest)</li> <li>Allows examination of individual cells</li> <li>Microscopy is the only method that can confirm the correct localization of your protein of interest after transfection</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Real-Time qPCR <p><a href="/evportal/destination/solutions?catID=LUSO4W8UU">Real-time qPCR</a> can quantify the expression level of a transgene or measure the degree to which a gene silencing (knockdown) experiment has been effective. It is fast and, because it requires little starting material, it is applicable when transfected cells are available in limited amounts.</p> <p>Using real-time qPCR, one can determine the relative abundance of specific messenger RNA (mRNA) compared to a control. Additionally, real-time qPCR can be used to assess how the knockdown of one gene may affect the expression of other genes in the pathway. However, it does not directly measure or identify the presence of protein. Therefore, any problems post-mRNA synthesis, such as problems during protein translation, will not be identified with this method. As a result, you may observe mRNA production where there may not be any protein synthesis.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Real-time PCR system needed</li> <li>Protein is not directly measured or identified</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Requires little starting material</li> <li>Commonly used and widely accepted technique</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Spectrometry <p>A spectrophotometer is an instrument that measures light intensity as a function of color or wavelength. In fact, &beta;-galactosidase assays can be used to determine transfection efficiency by measuring colorimetric changes associated with &beta;-galactosidase activity.</p> <p>For more information about spectrophotometry and &beta;-galactosidase assays, see:</p> <ul> <li>Nielsen DA et al. (1983). Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci USA 80, 5198&ndash;5202.</li> <li>Sambrook J, Fritsch EF, Maniatis T, eds. (1989). Molecular Cloning: A Laboratory Manual, Second Ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press).</li> <li><a href="http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240" target="_blank">http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240</a></li> </ul> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Expression of &beta;-galactosidase (when &beta;-galactosidase assays are used)</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate with spectrophotometer after addition of substrate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Western Blot Analysis <p><a href="/evportal/destination/solutions?catID=LUSPPAKG4">Western blot analysis</a> can be used to identify or quantitate the total expression of your transfected gene from a population of cells. With western blot analysis, you can determine the relative expression level of your transfected protein in a population of cells. This method of quantitation requires the use of an internal control, such as GAPDH or &beta;-actin.</p> <p>For more information about western blot analysis, see:</p> <ul> <li>Bio-Rad bulletin <a href="http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5723.pdf" target="_blank">5723</a>. Increase Western Blot Throughput With Multiplex Fluorescent Detection.</li> <li><a href="http://www.westernblotting.org/" target="_blank">http://www.westernblotting.org/</a></li> </ul> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Must be able to label and detect protein</li> <li>Time consuming due to running and transfer of gels and labeling protein for detection</li> <li>For quantitative analysis, need an internal control and densitometer</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Inexpensive</li> <li>Commonly used and widely accepted technique</li> <li>Can be used for quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> References <p><strong> General Transfection</strong></p> <p>Jordan E et al. (2007). Optimization of electroporation conditions with the Gene Pulser MXcell&trade; electroporation system. Bio-Rad bulletin 5622.</p> <p>Nickoloff JA, ed. (1995). Animal Cell Electroporation and Electrofusion Protocols (Methods in Molecular Biology, volume 48) (Totowa, NJ: Humana Press Inc.).</p> <p><strong> Flow Cytometry </strong></p> <p>SourceForge, Inc. (2007). Bioinformatics Standards for Flow Cytometry. <a href="http://flowcyt.sourceforge.net/" target="_blank">http://flowcyt.sourceforge.net/</a>, accessed August 4, 2009.</p> <p><strong> Fluorometry </strong></p> <p>Wilson J and Segal A (1998). Measuring intracellular enhanced green fluorescent protein with the VersaFluor&trade; fluorometer. Bio-Rad bulletin 2368.</p> <p><strong> Laser-Scanning Molecular Imaging </strong></p> <p>Bio-Rad bulletin 2752. Applications for Molecular Imager FX systems: Instrument settings.</p> <p><strong> Luminometry </strong></p> <p>Sambrook J and Russell D (2001). Analysis of gene expression in cultured mammalian cells. In Molecular Cloning: A Laboratory Manual, 3rd ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press), 17.96.</p> <p><strong> Microscopy </strong></p> <p>MC Services. Fluorescence Imaging. <a href="http://www.mwrn.com/microscopy/light/fluorescence.aspx" target="_blank">http://www.mwrn.com/microscopy/light/fluorescence.aspx</a>, accessed August 4, 2009.</p> <p>Rogers S. Cell Biology Applications of Fluorescence Microscopy. IHC World, LLC. <a href="http://www.ihcworld.com/_protocols/immunofluorescence/immunofluorescence.htm" target="_blank">http://www.ihcworld.com/_protocols/immunofluorescence/immunofluorescence.htm</a>, accessed August 4, 2009.</p> <p><strong> Real-Time Quantitative PCR </strong></p> <p>Bio-Rad Bulletin 5279. Real-time PCR applications guide.</p> <p><strong> Spectrophotometry </strong></p> <p>Nielsen DA et al. (1983). Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci USA 80, 5198&ndash;5202.<br /> Protocol Online. B-Gal Staining of Eukaryotic Cells in Vitro. <a href="http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240" target="_blank">http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240</a>, accessed August 4, 2009.</p> <p>Sambrook J, Fritsch EF, Maniatis T, eds. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press).</p> <p><strong> Western Blot Analysis</strong></p> <p>Bio-Rad bulletin 5723. Increase western blot throughput with multiplex fluorescent detection.<br /> westernblotting.org. <a href="http://www.westernblotting.org/" target="_blank">http://www.westernblotting.org/</a>, accessed August 4, 2009.</p> <div class="top"><a href="#helptop">Back to Top</a></div> <div class="videowrap"> <div class="videoImg"><a title="Cell Counting and Transfection" href="http://www.jove.com/video/1904/using-an-automated-cell-counter-to-simplify-gene-expression-studies-sirna-knockdown-of-il-4-dependent-gene-expression-in-namalwa-cells" target="_blank"><img src="/webroot/web/images/lsr/support/tutorials/global/ov_cell_counting_transfection.jpg" alt="Cell Counting and Transfection" /></a></div> <div class="videoDesc"><a title="Cell Counting and Transfection" href="http://www.jove.com/video/1904/using-an-automated-cell-counter-to-simplify-gene-expression-studies-sirna-knockdown-of-il-4-dependent-gene-expression-in-namalwa-cells" target="_blank">Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells</a></div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="Gene Pulser MXcell" href="http://www.jove.com/video/1662/using-the-gene-pulser-mxcell-electroporation-system-to-transfect-primary-cells-with-high-efficiency" target="_blank"><img src="/webroot/web/images/lsr/support/tutorials/global/ov_gene_pulser_mxcell.jpg" alt="Gene Pulser MXcell" /></a></div> <div class="videoDesc"><a title="Gene Pulser MXcell" href="http://www.jove.com/video/1662/using-the-gene-pulser-mxcell-electroporation-system-to-transfect-primary-cells-with-high-efficiency" target="_blank">Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency</a></div> <div class="clear">&nbsp;</div> </div> <div class="videowrap vwrap_last"> <div class="videoImg"><a title="Helios Gene Gun" href="http://www.jove.com/Details.stp?ID=675" target="_blank"><img src="/webroot/web/images/lsr/support/tutorials/global/helios_gene_gun.jpg" alt="Helios Gene Gun" /></a></div> <div class="videoDesc"><a title="Helios Gene Gun" href="http://www.jove.com/Details.stp?ID=675" target="_blank">Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture</a></div> <div class="clear">&nbsp;</div> </div> 5929 TC10 Automated Cell Counter Brochure, Rev B 5929 /webroot/web/pdf/lsr/literature/Bulletin 5929.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No TC10 Automated Cell Counter Brochure, Rev B 5929 5542 5542 Electroporation Systems Brochure, Rev A H /webroot/web/pdf/lsr/literature/Bulletin_5542.pdf Literature PDF Brochures and Specifications /webroot/web/images/general/icons/icon_pdf.gif No Electroporation Systems Brochure, Rev A Life Science 5542 165-2092, 165-2660, 165-2662, cell, cuvette, gene-pulser, genepulser, mammalian, shockpod, 165-2666, 1652091, 1652661, acids, arc-quenching, arq, electroporating, eukaryotic, fungal, micropulser, prokaryotic, transfect, 165-2082, 165-2091, 1652081, 1652083, 1652092, 1652100, 1652667, 1652669, arc quenching, ark, cuvet, cuvettes, Escherichia coli, eucaryotic, fungi, micro-pulse, shock-pod, 165-2081, 165-2086, 165-2093, 165-2661, 1652089, bacteria, cells, e., e.coli, electroporator, micropulse, pulsetrac, yeasts, 165-2083, 165-2100, 1652082, 1652093, 1652668, bacterial, dna, Gene-pulser xcell, pulse trac, rna, transform, yeast, 165-2089, 165-2667, 165-2669, 1652086, 1652088, 1652666, electroporators, fungus, saccharomyces cerevisiae, transfirmation, 165-2088, 165-2668, electroporaters, eucaryotes, eukaryotes, excel, gene transfer, prokaryotes, 1652660, 1652662, cuvets, electroporater, LIT5542, micro pulser, nucleic acid, track, transfection, transformation 5443 Biolistic Particle Delivery Systems Brochure, Rev A 5443 /webroot/web/pdf/lsr/literature/Bulletin_5443.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No Biolistic Particle Delivery Systems Brochure, Rev A 5443 165-2421, 165-2422, 1652422, cell-culture, chloroplasts, genes, Hepta adaptor, LIT5443, micro-carrier, micro-carriers, microparticle, plant, transfect, transfer, 165-2225, 165-2257, 165-2326, 165-2335, 1652225, 1652257, 1652278, 1652335, 1652432, bombardment, co-delivery, embryos, microcarrier, 1000/He, 165-2278, 1652326, 1652424, 1652431, bacteria, Helios Gene Gun System, mitochondrial, plasmid, transfecting, 165-2259, 165-2262, 165-2411, 165-2413, 165-2432, 1652413, bacterial, cotransformation, deoxyribonucleic, DNA, micro-particle, mitochondrion, PDS-1000/He, RNA, transform, yeast, 165-2258, 165-2336, 165-2412, 1652258, 1652336, cell culture, goldcoat, in vivo transformation, microcarriers, plasmids, 165-2431, 1652411, 1652418, acid-coated, bombard, chloroplast, micro-particles, rupture disks, 1652259, 1652421, co-transformation, codelivery, embryo, mitochondria, nucleic acid coated, ribonucleic, transfection, 165-2418, 165-2424, 1652262, 1652412, acceleration, animal, Heleos, helium pulse, microparticles, PDS 1000 He, pollen, trypsinized 2873 TransFectin Lipid Reagent Brochure, Rev A 2873 /webroot/web/pdf/lsr/literature/Bulletin_2873.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No TransFectin Lipid Reagent Brochure, Rev A 2873 LIT2873, gene transfer, bulletin 2873, transfection 3105 siLentFect Lipid Reagent Flier, Rev B 3105 /webroot/web/pdf/lsr/literature/Bulletin_3105B.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No siLentFect Lipid Reagent Flier, Rev B 3105 RNAi, bulletin 3105, LIT3105, Transfection 6039 5976 Gel Doc™ EZ Imager Brochure, Ver G 5976 H /webroot/web/pdf/lsr/literature/Bulletin_5976.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No Gel Doc EZ Imager Brochure 5976 imaging electrophoresis, high-quality, tray, green button, uv, ultraviolet, white, blue, stain-free, stain free, ethidium bromide, sybr, oriole fluorescent gel, gelred, sypro ruby, coomassie, fluor orange, krypton stain, stains, copper, silver, zinc, gelgreen, green, safe, gold, mini-protean tgx gels, criterion, western blot, blots, traditional sds-page, geldoc, image lab software, 1708270, 1708271, 1708272, 1708273, 1708274, 1708276, 1709690, 1707581, 1708089, 1708097, 4568023, 4568024, 4568026, 4568021, 4568025, 4568029, 4568033, 4568034, 4568036, 4568031, 4568035, 4568039, 4568043, 4568044, 4568046, 4568041, 4568045, 4568049, 4568083, 4568084, 4568086, 4568081, 4568085, 4568089, 4568093, 4568094, 4568096, 4568091, 4568095, 4568099, 4568103, 4568104, 4568106, 4568101, 4568105, 4568109, 4568123, 4568124, 4568126, 4568121, 4568125, 4568129, 5678023, 5678024, 5678025, 5678033, 5678034, 5678035, 5678043, 5678044, 5678045, 5678073, 5678074, 5678075, 5678072, 5678071, 5678083, 5678084, 5678085, 5678082, 5678081, 5678093, 5678094, 5678095, 5678092, 5678091, 5678103, 5678104, 5678105, 5678102, 5678101, 5678113, 5678114, 5678115, 5678112, 5678111, 5678123, 5678124, 5678121, 170-8270 5553 Gene Modulation Workflow Brochure, Rev B 5553 /webroot/web/pdf/lsr/literature/Bulletin_5553B.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No Gene Modulation Workflow Brochure, Rev B 5553 165-4000, 165-8000, 1652100, 170-8170, 1703940, 7007001, 732-6100, amplification, biolistics, CFD-3121, expression, GenePulser X-cell, multiplexing, PDS 1000-He, QuantumPrep, RT-PCR, sequencing, 1632086, 1632130, 1652431, 170-3351, 1709780, 1749950, 7326100, 7326343, BioOdyssey Calligrapher miniarrayer, CFB3120, interaction, Mini-PROTEAN Tetra cell, multi-color, PDS1000/He, PROTEAN IEF, quantification, quantitation, Ready Prep 2D, ReadyPrep 2-D cleanup kit, silent-mer dicer-substrate, silentfect RNAi, 165-3860, 165-3863, 1653861, 1653862, 1658000, 170-3940, 1703930, 1708170, 171-000201, 171-000205, 174-9950, 700-7000, 7007002, 732-6800, aqua-pure, blot, Gene Pulser Xcell total system, iso-electric, lipid mediated, microarray analysis, microarrays, mini-arrayer, multiplex suspension array technology, nucleic acid purification, reverse transcription, reverse-transcription, silent-fect, transfect, 165-2431, 165-2660, 170-3930, 1703360, Aqua, arrayer, CFD3121, electroporate, Gel-doc, Helios gun, iQ5 multicolor real-time PCR detection, MiniProtean, multi-plex, ProteOn XPR36, Quantum, real time polymerase chain reaction, sequigen, trans, Zeta-Probe membranes, 165-2257, 1652088, 1652257, 1652432, 169-2000, 1692000, 1692100, 170-9780, 1703351, 1708171, 1709300, 171000205, 1760100, 732-6340, Aurum, blotter, chemical transformation, cyclers, lipid-mediated, LIT5553, Molecular Imager Gel Doc XR, Quantum-prep plasmid miniprep, ribo-nucleic, sequi-gen GT, 1000 He, 163-2130, 165-2088, 165-3862, 1653863, 169-2300, 7326800, arrays, bioplex, blotting, CFB-3120, phenotype, Prote-on XPR-36, silentmer validated dicer substrate siRNA duplexes, thermal cycler, 1645050, 165-2100, 165-2432, 169-2100, 169-2200, 170-8171, 170-9300, 171000201, 7007000, 732-6343, Bio-Plex 200, biolistic particle delivery, deoxy-ribo, deoxyribonucleic, DNA transfer, Experion automated electrophoresis, geldoc, image, isoelectric focusing, mini-opticon, MiniOpticon, PDS-1000/He, protein profiling, RNA, TransBlot-SD, transform, Zeta Probe membrane, 163-2086, 164-5050, 165-3861, 1652660, 1653860, 1654000, 1692200, 1692300, 170-3360, 176-0100, 700-7001, 700-7002, 7326340, Aquapure genomic, electroporation, Micro-pulser cuvettes, Micropulser electroporator, mini-prep, multiplexed, PROTEON, Ready-Prep 2 D, ribonucleic, Trans-blot SD semi-dry, trans-fectin, Transfectin, transfection, XPR 36 5924 Stem Cell Basics for Life Science Researchers Brochure, Rev A 5924 H /webroot/web/pdf/lsr/literature/Bulletin_5924A.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/icon_pdf.gif No Stem Cell Basics for Life Science Researchers Brochure, Rev A 5924 Bulletin 5924, embryonic differentiation, embryo, isolated pluripotent sc, development, multipotent, unipotent, terminally differentiated, blastocyst, mass of inner cells, ICM, es, history, hes, mes, human, mouse, lines, adult, somatic, imprinting, chimeric, programming, reprogramming, ips, isolation and maintenance, growth factors, media, medium, tissue culture, fibroblast, fibroblasts, feeder layer, hematopoietic, induced, ectoderm, mesoderm, endoderm, transfection, knock down, RNAi, viral-mediated delivery, lipid-mediated, electroporation, biolistic particle, retrovirus, genomic analysis, proteomic, expression, phenotype, karotyping, SNPs, SNP, PCR, microarray, profiling, profile, qPCR, array, flow cytometry, immunocytochemistry, RT-PCR, RT-qPCR, western blotting, Bio-Plex suspension array system, sso7d fusion protein technology, 170-8891, 170-8893, 170-8895, 170-8899, 172-5203, 170-8885, 172-5213, 172-5103, 172-5853, 172-5233, 170-8864, 172-5849, 172-5253, 172-5243, 172-5108, 172-5857, 165-2661, 165-2670, 165-2674, 165-2677, 165-2681, 165-2081, 165-2082, 165-2086, 165-2088, 165-2091, 165-2092, 170-8201, 170-8351, 170-8202, 170-8352, 170-8206, 170-8353, 170-8203, 170-8354, 170-8204, 170-8205, 170-8200, 1708891, 1708893, 1708895, 1708899, 1725203, 1708885, 1725213, 1725103, 1725853, 1725233, 1708864, 1725849, 1725253, 1725243, 1725108, 1725857, 1652661, 1652670, 1652674, 1652677, 1652681, 1652081, 1652082, 1652086, 1652088, 1652091, 1652092, 1708201, 1708351, 1708202, 1708352, 1708206, 1708353, 1708203, 1708354, 1708204, 1708205, 1708200 5554 Gene Silencing | RNAi Workflow Brochure, Rev B 5554 /webroot/web/pdf/lsr/literature/Bulletin_5554.pdf Literature PDF Brochures_and_Specifications /webroot/web/images/general/icons/icon_pdf.gif No Gene Silencing | RNAi Workflow Brochure, Rev B 5554 1610449, 171-000201, 174-9974, 174-9975, 7007102, 732-6800, Aqua-pure, automated electrophoresis, blot, iso-electric, microarrays, microinjection, micropulser, mRNA quantification, multiplex suspension array technology, reverse transcription, reverse-transcription, RNA interference, silent-mer predesigned, small inhibitory RNAs, transfect, 1000, 161-0737, 163-2105, 1632130, 1652431, 1709780, 174-9970, 1749950, 700-7106, 7326370, BioOdyssey Calligrapher miniarrayer, micro-array, Mini-PROTEAN Tetra cell, multi-color, plasmid, ReadyPrep 2-D cleanup, ribonucleic acid, silentfect reagent, 165-4000, 1703350, 174-9960, 1749975, 1749977, 732-6370, 74-9950, amplification, Bio-Odyssey mini-arrayer, Laemmli buffer, multi-plexed, multi-plexing, multiplexing, PDS-1000 He, RT-PCR, spotting, transfected, vector-mediated, 165-2432, 170-3350, 171000201, 174-9961, 1749974, 1749976, 700-7102, 732-6890, Bio-Plex 200, biolistic particle delivery, Experion, Gene-Pulser Xcell total system, isoelectric focusing, micro-injection, micro-pulser, PDS-1000/He, Pure-Zol, validated siRNAs, 1610737, 165-2257, 1652088, 1652257, 1652432, 169-2000, 1692000, 170-9780, 174-9971, 1749971, blotter, cyclers, delivery optimization kit, IEF, qPCR, RT-qPCR, shRNA, 161-0449, 165-2431, 165-2660, 1703360, 1749961, 1749973, 7007106, aqua pure, arrayer, Bio Plex, chemically-mediated, genomics, Helios gun, iQ5 multicolor detection, Mini PROTEAN, multi-plex, nucleic, proteomics, Ready Prep 2 D, Real time polymerase chain reaction, Zol, 163-2130, 165-2088, 174-9972, 174-9977, 1749960, 1749970, 1749972, 7326800, 7326890, Aquapure isolation, arrays, BioPlex, chemically mediated, lipid transfection, LIT5554, PDS 1000/He, PureZol reagent, shRNAs, silentmer dicer-substrate siRNA duplexes, thermal cycler, 161-0490, 1610490, 1632105, 1652660, 1654000, 170-3360, 174-9973, 174-9976, 700-7104, 7007104, Aurum 96, electroporation, GenePulser cuvettes, microarray, multiplexed, Protein expression analysis, PROTEON, ready-prep 2D, real-time PCR, short hairpin RNAs, specific inhibition, thermal-cyclers, transfectin, western blotting 5969 Post Transfection Analysis 5969 /webroot/web/pdf/lsr/literature/Bulletin_5969.pdf Literature PDF Other /webroot/web/images/general/icons/icon_pdf.gif No Post Transfection Analysis 5969 5970 Basic Cell Culture 5970 /webroot/web/pdf/lsr/literature/Bulletin_5970.pdf Literature PDF Other /webroot/web/images/general/icons/icon_pdf.gif Basic Cell Culture No An introduction to growing and maintaining mammalian cells in culture. 5970 cell culture, mammalian, tissue, zoe, fluorescent, imager, proliferation, organelle, staining, tc10, tc20, counter, counting, sorter, sorting, flow, cytometry, 145-1005, 145-1006, 145-1008, 145-0102, 145-0031, 166-0476, 166-0476EDU, 1451005, 1451006, 1451008, 1450102, 1450031, 1660476, 1660476EDU Life Science Research/Products/Transfection/Electroporation/Gene Pulser Xcell Electroporation Systems ->MT::b1a35eb3-d55c-47b3-aaf3-95e4d1d85848##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Semi-Dry Blotting Systems/Trans-Blot Turbo Transfer System ->MTS::LGOQBW15##Life Science Research/Products/Amplification - PCR/Real-Time PCR Detection Systems/CFX96 Touch Real-Time PCR Detection System ->MTS::LJB1YU15##Life Science Research/Products/Sample Quantitation/Fluorometry/VersaFluor Fluorometer ->MT::b100e565-eb31-4303-8305-9db89ddcf920##Life Science Research/Products/Imaging Instruments &amp; Bioinformatics/Molecular Imager Systems/Gel Doc EZ Systems ->MTS::L7BL4S15## Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR ->MTS::LUSO4W8UU##Life Science Research/Solutions/Technologies/Western Blotting ->MTS::LUSPPAKG4##Life Science Research/Solutions/Technologies/Imaging and Analysis/Imaging Systems ->MTS::LUSQCPKSY##Life Science Research/Solutions/Technologies/Cell Counting Methods ->MTS::LUSOLB470## Eddie C Posttransfection Analysis of Cells 12/29/11 03:43 PM 12/29/21 03:44 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Technologies/Transfection N 0 Introduction to Transfection /en-us/applications-technologies/applications-technologies/posttransfection-analysis-cells?ID=LUSONCB9O

This section describes different methods of analyzing cells after transfection, such as flow cytometry, fluorometry, laser-scanning molecular imaging, luminometry, real-time qPCR, microscopy, spectrometry, and western blot analysis. It also provides guidelines and methods that you can use for the analysis of transfected cells. The methods can be used to determine transfection efficiencies and to perform more in-depth analyses of the expression of your favorite gene/protein. However, depending on your cells, reagents, or equipment availability these methods should be modified to fit your laboratory needs.

Related Topics: Transfection, Instrument-Based Transfection Methods, and Chemical- and Viral-Based Transfection.

 

General Considerations

There are many factors that can influence transfection efficiency, a number of which are specific to the target cell. Cell-related factors affecting transfection include cell density, cell size, replication state, passage number, health of cells, biomolecule type, and concentration. Some factors are method specific; for example, electroporation parameters such as voltage, capacitance, and resistance strongly affect transfection efficiency. Therefore, to obtain high efficiencies, all relevant factors should be considered when planning transfection experiments.

Frequently, after any transfection experiment, it is important to assess the efficiency of transfection and the impact of transfection on the cells. Analysis of transfection efficiency can be as simple as confirming the expression of your gene of interest. However, in many cases assessment of transfection involves determining the total expression level of your gene of interest, determining the number of positive cells within a transfected population (% positive cells), and/or visual confirmation of your protein of interest (Jordan et al. 2007).

Most methods for measuring protein expression level of your transfected cell will determine the total expression from a population of transfected cells. Measurement of total gene expression can be done through real-time quantitative PCR (real-time qPCR), western blot analysis, molecular imaging, and fluorometry. Determining the number of positive cells within a transfected cell population can be performed by microscopy and flow cytometry. Finally, confirming localization of your protein of interest can be done by microscopy.

Reporter genes, such as green fluorescent protein (GFP), luciferase, or β-galactosidase can be used to analyze transfection efficiency because their expression can be easily monitored. They can also be used to standardize transfection efficiencies between different transfection experiments by comparing the expression levels of their products. Reporter genes can be used alone or fused to a gene of interest to determine the protein expression level, the number of positive cells, or the location of the protein being studied.

Following are some of the most common methods used for posttransfection analysis.

 

Flow Cytometry

A flow cytometer can determine the number of positive cells within a transfected cell population (% positive cells). In addition, a flow cytometer with sorting abilities can enrich for positive cell populations. However, flow cytometry requires that the cells either express a fluorescent protein, such as GFP, or your protein of interest must be labeled with a fluorescent molecule.

For more information about flow cytometry, visit http://flowcyt.sourceforge.net/.

Requirements or Limitations

  • Expensive
  • Can only detect fluorescence (often requires labeling protein with a fluorescent molecule)
  • Time consuming

Advantages

  • Determines percentage of positive cells (quantitative measurement)
  • Can enrich for positive cell population with sorting ability
 

Fluorometry

A fluorometer can detect a wide range of fluorescence. Extracts from cells expressing fluorescent protein can be used to measure the expression level of your gene in the transfected cells.

For more information about fluorescent protein detection in cell extracts, see Wilson J and Segal A (1998). Measuring intracellular enhanced green fluorescent protein with the VersaFluor™ fluorometer. Bio-Rad bulletin 2368.

Requirements or Limitations

  • Moderately expensive
  • Can only detect a fluorescent molecule or protein

Advantages

  • Able to perform a quick and easy sample detection (direct analysis of lysate)
  • Can perform quantitative analysis for total expression

A fluorometer can detect a wide range of fluorescence. Extracts from cells expressing fluorescent protein can be used to measure the expression level of your gene in the transfected cells.

For more information about fluorescent protein detection in cell extracts, see Wilson J and Segal A (1998). Measuring intracellular enhanced green fluorescent protein with the VersaFluor™ fluorometer. Bio-Rad bulletin 2368.

Requirements or Limitations

  • Moderately expensive
  • Can only detect a fluorescent molecule or protein

Advantages

  • Able to perform a quick and easy sample detection (direct analysis of lysate)
  • Can perform quantitative analysis for total expression
 

Laser-Scanning Molecular Imaging

Laser-scanning molecular imagers can detect a wide range of fluorescence. With the use of appropriate fluorophores, western blots or gels can be quantitatively analyzed with a scanner. In addition to analyzing western blots or gels, scanners can also directly detect and quantify lysate from a population of cells expressing a fluorescent protein, such as GFP. The ability to detect fluorescence allows the scanner to directly analyze cell lysate without running a gel.

For more information about laser-scanning molecular imaging, see Bio-Rad bulletin 2752. Applications for Molecular Imager FX Systems: Instrument settings.

Requirements or Limitations

  • Expensive
  • Can only detect a fluorescent molecule or protein
  • For quantitative analysis, need an internal control and densitometer

Advantages

  • Able to perform a quick and easy sample detection (direct analysis of lysate)
  • Can perform quantitative analysis for total expression
 

Luminometry

A luminometer is an instrument that measures light intensity. Expression or co-expression of luciferase, which emits light after interacting with its substrate, can be used for quantitative analysis of total protein expression.

For more information about luminometry and luciferase assays, see Sambrook J and Russell D (2001). Analysis of gene expression in cultured mammalian cells. In Molecular Cloning: A laboratory manual, Third Ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press), 17.96.

Requirements or Limitations

  • Moderately expensive
  • Luminometer needed
  • Requires expression of luciferase gene (when luciferase assays are used)
  • Only measures total gene expression from population of cells

Advantages

  • Able to perform a quick and easy sample detection (direct analysis of lysate with luminometer after addition of substrate)
  • Can perform quantitative analysis for total expression
 

Microscopy

Microscopy allows direct visualization of transfected cells. Microscopy can be used to count the number of transfected cells for estimation of positive cell numbers. Microscopy can also be used to confirm the correct localization of your protein of interest. Visualization of cells transfected with your gene of interest can be accomplished in several ways. Expression or co-expression of a fluorescent protein, such as GFP, will allow the direct identification of your transfected cells. However, a non-fluorescent protein must be labeled for visualization. Immunofluorescence, chemical labeling, and immunohistochemistry techniques can be used to label proteins. In general, microscopy cannot be used for quantitative measurement of transfection efficiency unless you have image analysis software that can count and quantitate positively transfected cells.

For more information about chemical and immunofluorescent labeling, fluorescence microscopy, and immunohistochemistry techniques, visit:

Requirements or Limitations

  • Expensive
  • Epifluorence microscope needed
  • Must be able to visualize protein (often requires labeling protein)
  • Time consuming if labeling protein is required

Advantages

  • Visual (direct observation of your protein of interest)
  • Allows examination of individual cells
  • Microscopy is the only method that can confirm the correct localization of your protein of interest after transfection
 

Real-Time qPCR

Real-time qPCR can quantify the expression level of a transgene or measure the degree to which a gene silencing (knockdown) experiment has been effective. It is fast and, because it requires little starting material, it is applicable when transfected cells are available in limited amounts.

Using real-time qPCR, one can determine the relative abundance of specific messenger RNA (mRNA) compared to a control. Additionally, real-time qPCR can be used to assess how the knockdown of one gene may affect the expression of other genes in the pathway. However, it does not directly measure or identify the presence of protein. Therefore, any problems post-mRNA synthesis, such as problems during protein translation, will not be identified with this method. As a result, you may observe mRNA production where there may not be any protein synthesis.

Requirements or Limitations

  • Moderately expensive
  • Real-time PCR system needed
  • Protein is not directly measured or identified

Advantages

  • Requires little starting material
  • Commonly used and widely accepted technique
 

Spectrometry

A spectrophotometer is an instrument that measures light intensity as a function of color or wavelength. In fact, β-galactosidase assays can be used to determine transfection efficiency by measuring colorimetric changes associated with β-galactosidase activity.

For more information about spectrophotometry and β-galactosidase assays, see:

  • Nielsen DA et al. (1983). Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci USA 80, 5198–5202.
  • Sambrook J, Fritsch EF, Maniatis T, eds. (1989). Molecular Cloning: A Laboratory Manual, Second Ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press).
  • http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240

Requirements or Limitations

  • Moderately expensive
  • Expression of β-galactosidase (when β-galactosidase assays are used)

Advantages

  • Able to perform a quick and easy sample detection (direct analysis of lysate with spectrophotometer after addition of substrate)
  • Can perform quantitative analysis for total expression
 

Western Blot Analysis

Western blot analysis can be used to identify or quantitate the total expression of your transfected gene from a population of cells. With western blot analysis, you can determine the relative expression level of your transfected protein in a population of cells. This method of quantitation requires the use of an internal control, such as GAPDH or β-actin.

For more information about western blot analysis, see:

Requirements or Limitations

  • Must be able to label and detect protein
  • Time consuming due to running and transfer of gels and labeling protein for detection
  • For quantitative analysis, need an internal control and densitometer

Advantages

  • Inexpensive
  • Commonly used and widely accepted technique
  • Can be used for quantitative analysis for total expression
 

References

General Transfection

Jordan E et al. (2007). Optimization of electroporation conditions with the Gene Pulser MXcell™ electroporation system. Bio-Rad bulletin 5622.

Nickoloff JA, ed. (1995). Animal Cell Electroporation and Electrofusion Protocols (Methods in Molecular Biology, volume 48) (Totowa, NJ: Humana Press Inc.).

Flow Cytometry

SourceForge, Inc. (2007). Bioinformatics Standards for Flow Cytometry. http://flowcyt.sourceforge.net/, accessed August 4, 2009.

Fluorometry

Wilson J and Segal A (1998). Measuring intracellular enhanced green fluorescent protein with the VersaFluor™ fluorometer. Bio-Rad bulletin 2368.

Laser-Scanning Molecular Imaging

Bio-Rad bulletin 2752. Applications for Molecular Imager FX systems: Instrument settings.

Luminometry

Sambrook J and Russell D (2001). Analysis of gene expression in cultured mammalian cells. In Molecular Cloning: A Laboratory Manual, 3rd ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press), 17.96.

Microscopy

MC Services. Fluorescence Imaging. http://www.mwrn.com/microscopy/light/fluorescence.aspx, accessed August 4, 2009.

Rogers S. Cell Biology Applications of Fluorescence Microscopy. IHC World, LLC. http://www.ihcworld.com/_protocols/immunofluorescence/immunofluorescence.htm, accessed August 4, 2009.

Real-Time Quantitative PCR

Bio-Rad Bulletin 5279. Real-time PCR applications guide.

Spectrophotometry

Nielsen DA et al. (1983). Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci USA 80, 5198–5202.
Protocol Online. B-Gal Staining of Eukaryotic Cells in Vitro. http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240, accessed August 4, 2009.

Sambrook J, Fritsch EF, Maniatis T, eds. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press).

Western Blot Analysis

Bio-Rad bulletin 5723. Increase western blot throughput with multiplex fluorescent detection.
westernblotting.org. http://www.westernblotting.org/, accessed August 4, 2009.

 

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3105 siLentFect Lipid Reagent Flier, Rev B Click to download
5976 Gel Doc™ EZ Imager Brochure, Ver G Click to download
5553 Gene Modulation Workflow Brochure, Rev B Click to download
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5554 Gene Silencing | RNAi Workflow Brochure, Rev B Click to download
5969 Post Transfection Analysis Click to download
5970 Basic Cell Culture Click to download
 
 
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It also provides guidelines and methods that you can use for the analysis of transfected cells. The methods can be used to determine transfection efficiencies and to perform more in-depth analyses of the expression of your favorite gene/protein. However, depending on your cells, reagents, or equipment availability these methods should be modified to fit your laboratory needs.</p> <p><strong>Related Topics:</strong> <a href="/evportal/destination/solutions?catID=LUSONCB9O">Transfection,</a> <a href="/evportal/destination/solutions?catID=LUSONV30E">Instrument-Based Transfection Methods</a>, and <a href="/evportal/destination/solutions?catID=LUSOOP49">Chemical- and Viral-Based Transfection</a>.</p> General Considerations <p>There are many factors that can influence transfection efficiency, a number of which are specific to the target cell. Cell-related factors affecting transfection include cell density, cell size, replication state, passage number, health of cells, biomolecule type, and concentration. Some factors are method specific; for example, electroporation parameters such as voltage, capacitance, and resistance strongly affect transfection efficiency. Therefore, to obtain high efficiencies, all relevant factors should be considered when planning transfection experiments.</p> <p>Frequently, after any transfection experiment, it is important to assess the efficiency of transfection and the impact of transfection on the cells. Analysis of transfection efficiency can be as simple as confirming the expression of your gene of interest. However, in many cases assessment of transfection involves determining the total expression level of your gene of interest, determining the number of positive cells within a transfected population (% positive cells), and/or visual confirmation of your protein of interest (Jordan et al. 2007).</p> <p>Most methods for measuring protein expression level of your transfected cell will determine the total expression from a population of transfected cells. Measurement of total gene expression can be done through real-time quantitative PCR (real-time qPCR), western blot analysis, molecular imaging, and fluorometry. Determining the number of positive cells within a transfected cell population can be performed by microscopy and flow cytometry. Finally, confirming localization of your protein of interest can be done by microscopy.</p> <p>Reporter genes, such as green fluorescent protein (GFP), luciferase, or &beta;-galactosidase can be used to analyze transfection efficiency because their expression can be easily monitored. They can also be used to standardize transfection efficiencies between different transfection experiments by comparing the expression levels of their products. Reporter genes can be used alone or fused to a gene of interest to determine the protein expression level, the number of positive cells, or the location of the protein being studied.</p> <p>Following are some of the most common methods used for posttransfection analysis.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Flow Cytometry <p>A flow cytometer can determine the number of positive cells within a transfected cell population (% positive cells). In addition, a flow cytometer with sorting abilities can enrich for positive cell populations. However, flow cytometry requires that the cells either express a fluorescent protein, such as GFP, or your protein of interest must be labeled with a fluorescent molecule.</p> <p>For more information about flow cytometry, visit <a href="http://flowcyt.sourceforge.net/" target="_blank">http://flowcyt.sourceforge.net/</a>.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Expensive</li> <li>Can only detect fluorescence (often requires labeling protein with a fluorescent molecule)</li> <li>Time consuming</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Determines percentage of positive cells (quantitative measurement)</li> <li>Can enrich for positive cell population with sorting ability</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Fluorometry <p>A fluorometer can detect a wide range of fluorescence. Extracts from cells expressing fluorescent protein can be used to measure the expression level of your gene in the transfected cells.</p> <p>For more information about fluorescent protein detection in cell extracts, see Wilson J and Segal A (1998). Measuring intracellular enhanced green fluorescent protein with the VersaFluor&trade; fluorometer. Bio-Rad bulletin <a href="http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2368.pdf" target="_blank">2368</a>.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Can only detect a fluorescent molecule or protein</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <p>A fluorometer can detect a wide range of fluorescence. Extracts from cells expressing fluorescent protein can be used to measure the expression level of your gene in the transfected cells.</p> <p>For more information about fluorescent protein detection in cell extracts, see Wilson J and Segal A (1998). Measuring intracellular enhanced green fluorescent protein with the VersaFluor&trade; fluorometer. Bio-Rad bulletin <a href="http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2368.pdf" target="_blank">2368</a>.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Can only detect a fluorescent molecule or protein</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Laser-Scanning Molecular Imaging <p>Laser-scanning molecular imagers can detect a wide range of fluorescence. With the use of appropriate fluorophores, western blots or gels can be quantitatively analyzed with a scanner. In addition to analyzing western blots or gels, scanners can also directly detect and quantify lysate from a population of cells expressing a fluorescent protein, such as GFP. The ability to detect fluorescence allows the scanner to directly analyze cell lysate without running a gel.</p> <p>For more information about laser-scanning molecular imaging, see Bio-Rad bulletin <a href="http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2752.pdf" target="_blank">2752</a>. Applications for Molecular Imager FX Systems: Instrument settings.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Expensive</li> <li>Can only detect a fluorescent molecule or protein</li> <li>For quantitative analysis, need an internal control and densitometer</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Luminometry <p>A luminometer is an instrument that measures light intensity. Expression or co-expression of luciferase, which emits light after interacting with its substrate, can be used for quantitative analysis of total protein expression.</p> <p>For more information about luminometry and luciferase assays, see Sambrook J and Russell D (2001). Analysis of gene expression in cultured mammalian cells. In Molecular Cloning: A laboratory manual, Third Ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press), 17.96.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Luminometer needed</li> <li>Requires expression of luciferase gene (when luciferase assays are used)</li> <li>Only measures total gene expression from population of cells</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate with luminometer after addition of substrate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Microscopy <p>Microscopy allows direct visualization of transfected cells. Microscopy can be used to count the number of transfected cells for estimation of positive cell numbers. Microscopy can also be used to confirm the correct localization of your protein of interest. Visualization of cells transfected with your gene of interest can be accomplished in several ways. Expression or co-expression of a fluorescent protein, such as GFP, will allow the direct identification of your transfected cells. However, a non-fluorescent protein must be labeled for visualization. Immunofluorescence, chemical labeling, and immunohistochemistry techniques can be used to label proteins. In general, microscopy cannot be used for quantitative measurement of transfection efficiency unless you have image analysis software that can count and quantitate positively transfected cells.</p> <p>For more information about chemical and immunofluorescent labeling, fluorescence microscopy, and immunohistochemistry techniques, visit:</p> <ul> <li><a href="http://www.ihcworld.com/_protocols/immunofluorescence/immunofluorescence.htm" target="_blank">http://www.ihcworld.com/_protocols/immunofluorescence/immunofluorescence.htm</a></li> <li><a href="http://www.mwrn.com/microscopy/light/fluorescence.aspx" target="_blank">http://www.mwrn.com/microscopy/light/fluorescence.aspx</a></li> </ul> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Expensive</li> <li>Epifluorence microscope needed</li> <li>Must be able to visualize protein (often requires labeling protein)</li> <li>Time consuming if labeling protein is required</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Visual (direct observation of your protein of interest)</li> <li>Allows examination of individual cells</li> <li>Microscopy is the only method that can confirm the correct localization of your protein of interest after transfection</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Real-Time qPCR <p><a href="/evportal/destination/solutions?catID=LUSO4W8UU">Real-time qPCR</a> can quantify the expression level of a transgene or measure the degree to which a gene silencing (knockdown) experiment has been effective. It is fast and, because it requires little starting material, it is applicable when transfected cells are available in limited amounts.</p> <p>Using real-time qPCR, one can determine the relative abundance of specific messenger RNA (mRNA) compared to a control. Additionally, real-time qPCR can be used to assess how the knockdown of one gene may affect the expression of other genes in the pathway. However, it does not directly measure or identify the presence of protein. Therefore, any problems post-mRNA synthesis, such as problems during protein translation, will not be identified with this method. As a result, you may observe mRNA production where there may not be any protein synthesis.</p> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Real-time PCR system needed</li> <li>Protein is not directly measured or identified</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Requires little starting material</li> <li>Commonly used and widely accepted technique</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Spectrometry <p>A spectrophotometer is an instrument that measures light intensity as a function of color or wavelength. In fact, &beta;-galactosidase assays can be used to determine transfection efficiency by measuring colorimetric changes associated with &beta;-galactosidase activity.</p> <p>For more information about spectrophotometry and &beta;-galactosidase assays, see:</p> <ul> <li>Nielsen DA et al. (1983). Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci USA 80, 5198&ndash;5202.</li> <li>Sambrook J, Fritsch EF, Maniatis T, eds. (1989). Molecular Cloning: A Laboratory Manual, Second Ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press).</li> <li><a href="http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240" target="_blank">http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240</a></li> </ul> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Moderately expensive</li> <li>Expression of &beta;-galactosidase (when &beta;-galactosidase assays are used)</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Able to perform a quick and easy sample detection (direct analysis of lysate with spectrophotometer after addition of substrate)</li> <li>Can perform quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> Western Blot Analysis <p><a href="/evportal/destination/solutions?catID=LUSPPAKG4">Western blot analysis</a> can be used to identify or quantitate the total expression of your transfected gene from a population of cells. With western blot analysis, you can determine the relative expression level of your transfected protein in a population of cells. This method of quantitation requires the use of an internal control, such as GAPDH or &beta;-actin.</p> <p>For more information about western blot analysis, see:</p> <ul> <li>Bio-Rad bulletin <a href="http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5723.pdf" target="_blank">5723</a>. Increase Western Blot Throughput With Multiplex Fluorescent Detection.</li> <li><a href="http://www.westernblotting.org/" target="_blank">http://www.westernblotting.org/</a></li> </ul> <p><strong> Requirements or Limitations</strong></p> <ul> <li>Must be able to label and detect protein</li> <li>Time consuming due to running and transfer of gels and labeling protein for detection</li> <li>For quantitative analysis, need an internal control and densitometer</li> </ul> <p><strong> Advantages</strong></p> <ul> <li>Inexpensive</li> <li>Commonly used and widely accepted technique</li> <li>Can be used for quantitative analysis for total expression</li> </ul> <div class="top"><a href="#helptop">Back to Top</a></div> References <p><strong> General Transfection</strong></p> <p>Jordan E et al. (2007). Optimization of electroporation conditions with the Gene Pulser MXcell&trade; electroporation system. Bio-Rad bulletin 5622.</p> <p>Nickoloff JA, ed. (1995). Animal Cell Electroporation and Electrofusion Protocols (Methods in Molecular Biology, volume 48) (Totowa, NJ: Humana Press Inc.).</p> <p><strong> Flow Cytometry </strong></p> <p>SourceForge, Inc. (2007). Bioinformatics Standards for Flow Cytometry. <a href="http://flowcyt.sourceforge.net/" target="_blank">http://flowcyt.sourceforge.net/</a>, accessed August 4, 2009.</p> <p><strong> Fluorometry </strong></p> <p>Wilson J and Segal A (1998). Measuring intracellular enhanced green fluorescent protein with the VersaFluor&trade; fluorometer. Bio-Rad bulletin 2368.</p> <p><strong> Laser-Scanning Molecular Imaging </strong></p> <p>Bio-Rad bulletin 2752. Applications for Molecular Imager FX systems: Instrument settings.</p> <p><strong> Luminometry </strong></p> <p>Sambrook J and Russell D (2001). Analysis of gene expression in cultured mammalian cells. In Molecular Cloning: A Laboratory Manual, 3rd ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press), 17.96.</p> <p><strong> Microscopy </strong></p> <p>MC Services. Fluorescence Imaging. <a href="http://www.mwrn.com/microscopy/light/fluorescence.aspx" target="_blank">http://www.mwrn.com/microscopy/light/fluorescence.aspx</a>, accessed August 4, 2009.</p> <p>Rogers S. Cell Biology Applications of Fluorescence Microscopy. IHC World, LLC. <a href="http://www.ihcworld.com/_protocols/immunofluorescence/immunofluorescence.htm" target="_blank">http://www.ihcworld.com/_protocols/immunofluorescence/immunofluorescence.htm</a>, accessed August 4, 2009.</p> <p><strong> Real-Time Quantitative PCR </strong></p> <p>Bio-Rad Bulletin 5279. Real-time PCR applications guide.</p> <p><strong> Spectrophotometry </strong></p> <p>Nielsen DA et al. (1983). Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. Proc Natl Acad Sci USA 80, 5198&ndash;5202.<br /> Protocol Online. B-Gal Staining of Eukaryotic Cells in Vitro. <a href="http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240" target="_blank">http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1240</a>, accessed August 4, 2009.</p> <p>Sambrook J, Fritsch EF, Maniatis T, eds. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. (Woodbury, NY: Cold Spring Harbor Laboratory Press).</p> <p><strong> Western Blot Analysis</strong></p> <p>Bio-Rad bulletin 5723. Increase western blot throughput with multiplex fluorescent detection.<br /> westernblotting.org. <a href="http://www.westernblotting.org/" target="_blank">http://www.westernblotting.org/</a>, accessed August 4, 2009.</p> <div class="top"><a href="#helptop">Back to Top</a></div> <div class="videowrap"> <div class="videoImg"><a title="Cell Counting and Transfection" href="http://www.jove.com/video/1904/using-an-automated-cell-counter-to-simplify-gene-expression-studies-sirna-knockdown-of-il-4-dependent-gene-expression-in-namalwa-cells" target="_blank"><img src="/webroot/web/images/lsr/support/tutorials/global/ov_cell_counting_transfection.jpg" alt="Cell Counting and Transfection" /></a></div> <div class="videoDesc"><a title="Cell Counting and Transfection" href="http://www.jove.com/video/1904/using-an-automated-cell-counter-to-simplify-gene-expression-studies-sirna-knockdown-of-il-4-dependent-gene-expression-in-namalwa-cells" target="_blank">Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells</a></div> <div class="clear">&nbsp;</div> </div> <div class="videowrap"> <div class="videoImg"><a title="Gene Pulser MXcell" href="http://www.jove.com/video/1662/using-the-gene-pulser-mxcell-electroporation-system-to-transfect-primary-cells-with-high-efficiency" target="_blank"><img src="/webroot/web/images/lsr/support/tutorials/global/ov_gene_pulser_mxcell.jpg" alt="Gene Pulser MXcell" /></a></div> <div class="videoDesc"><a title="Gene Pulser MXcell" href="http://www.jove.com/video/1662/using-the-gene-pulser-mxcell-electroporation-system-to-transfect-primary-cells-with-high-efficiency" target="_blank">Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency</a></div> <div class="clear">&nbsp;</div> </div> <div class="videowrap vwrap_last"> <div class="videoImg"><a title="Helios Gene Gun" href="http://www.jove.com/Details.stp?ID=675" target="_blank"><img src="/webroot/web/images/lsr/support/tutorials/global/helios_gene_gun.jpg" alt="Helios Gene Gun" /></a></div> <div class="videoDesc"><a title="Helios Gene Gun" href="http://www.jove.com/Details.stp?ID=675" target="_blank">Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture</a></div> <div class="clear">&nbsp;</div> </div> 5929 5542 5443 2873 3105 6039 5976 5553 5924 5554 5969 5970 Life Science Research/Products/Transfection/Electroporation/Gene Pulser Xcell Electroporation Systems ->MT::b1a35eb3-d55c-47b3-aaf3-95e4d1d85848##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Semi-Dry Blotting Systems/Trans-Blot Turbo Transfer System ->MTS::LGOQBW15##Life Science Research/Products/Amplification - PCR/Real-Time PCR Detection Systems/CFX96 Touch Real-Time PCR Detection System ->MTS::LJB1YU15##Life Science Research/Products/Sample Quantitation/Fluorometry/VersaFluor Fluorometer ->MT::b100e565-eb31-4303-8305-9db89ddcf920##Life Science Research/Products/Imaging Instruments &amp; Bioinformatics/Molecular Imager Systems/Gel Doc EZ Systems ->MTS::L7BL4S15## Life Science Research/Solutions/Technologies/qPCR|Real-Time PCR ->MTS::LUSO4W8UU##Life Science Research/Solutions/Technologies/Western Blotting ->MTS::LUSPPAKG4##Life Science Research/Solutions/Technologies/Imaging and Analysis/Imaging Systems ->MTS::LUSQCPKSY##Life Science Research/Solutions/Technologies/Cell Counting Methods ->MTS::LUSOLB470## Eddie C Posttransfection Analysis of Cells 12/29/11 03:43 PM 12/29/21 03:44 PM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA en LSR /LSR/Technologies/Transfection N 0
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