Introduction
MicroRNAs have emerged as important regulators of gene expression that modulate the activity of many important biological processes. With more than 700 human miRNAs identified so far1, it is not surprising that mutations and improper regulation of miRNAs have been linked to a variety of physiological disorders such as cancer and heart disease2,3. Recent studies have shown that the majority of miRNA regulatory binding sites are found in 3'UTRs4,5, and some researchers have estimated that there are hundreds of targets per miRNA in the cell6. Given the biological importance of miRNAs, the large number of miRNAs that exist and the large number of targets for each of them, it is critical that researchers are able to reliably identify endogenous targets of miRNAs, preferably on platforms amenable to high-throughput studies.
While utilizing computational tools like miRBase, TargetScan, and PicTar1,6,7 is a powerful way to generate lists of putative mRNA targets for any miRNA, the predictions are not yet accurate enough to be relied upon alone8. Therefore, researchers also leverage a number of experimental approaches to identify targets de novo or to validate predicted targets. Many teams have relied on high-throughput measures of steady-state mRNA or protein levels to identify genes for which expression changes when miRNA activity is perturbed4,9,10. Such changes are undoubtedly biologically meaningful and represent an important first step, however measuring changes in overall expression does not distinguish between direct effects of a miRNA on a transcript and indirect or downstream signaling effects. Other powerful tools that provide evidence for direct miRNA-mRNA interactions are chromatin-immunoprecipitation (ChIP) strategies that cross-link argonaute proteins (and thus the RISC complex) to miRNAs and mRNAs, though identifying the interacting partners does not immediately indicate the functional effects of such a binding event5,11. Thus, 3'UTR-reporter assays that provide functional evidence for a miRNA's effect on a particular UTR have become an important component of a thorough miRNA study. Such cell-based assay data taken together with computational target predictions or expression and proteomics data provides strong evidence that a particular 3'UTR is directly regulated by the miRNA of interest. 
In a 3'UTR-reporter assay, the 3'UTR from a gene of interest is fused to the end of a luciferase reporter gene. If the 3'UTR is a target of a miRNA, the reporter transcript stability and/or its translation efficiency will change with variation in functional miRNA concentration. So unlike transcript-only based assays, reporter assays help identify effects on both steady state mRNA and protein levels. Reporter systems may also be used to study the functional effects of mutagenizing a putative miRNA binding site in a 3'UTR to confirm that it is indeed necessary for the interaction8. And while large-scale studies using expression arrays, proteomics techniques, and argonaute ChIP provide valuable genome-wide data for a small number of experimental conditions, cell-based reporter assays are the most amenable to scaling up for the study of miRNA-3'UTR interactions in hundreds or thousands of conditions as may become necessary for high-throughput screening of small molecules or other library-based effectors. 
Many scientists have applied reporter assay technology to the study of miRNA-3'UTR interactions. However, with the idea that many miRNAs each target hundreds of different transcripts, it has been difficult for researchers to utilize this powerful approach on a high-throughput scale because the cloning, validation, and optimization steps for creating thousands of different 3'UTR-reporter vectors are often cost prohibitive. In an effort to make small- and large-scale 3'UTR-reporter studies accessible to any researcher, we have created a genome-wide collection of human 3'UTRs pre-cloned into the highly-optimized LightSwitch Luciferase Assay System. The reporter system also includes validated control vectors, a set of more than 700 synthetic miRNA target reporter constructs for use as positive controls, and a series of standardized experimental protocols.
Using the following protocol, you can ask whether a particular human 3'UTR is a target of your miRNA of interest. The critical steps in this workflow are experimental design, transfection of reporter constructs and miRNA mimics, luciferase reporter assays, and data analysis. For experimental design, it is important thoughtfully select a transfectable cell line, experimental and control reporter constructs, and miRNA mimics and non-targeting controls. Common highly-transfectable adherent cell lines like HT1080, HepG2, HCT-116, Hela, 293, and others often work well with this protocol.
Important notes on experimental design
The LightSwitch Luciferase Assay System is a fully optimized reporter system that includes GoClone constructs utilizing the RenSP luciferase gene and LightSwitch Luciferase Assay Reagents. Use all components of the LightSwitch System as recommended to obtain optimal reporter assay results.
Co-transfection of any oligos/plasmids with SwitchGear GoClone reporter constructs reduces overall luciferase signals in a non-specific manner. Therefore, it is important to perform the following transfection combinations: reporter only, reporter + non-targeting control miRNA, and reporter + specific miRNA.
Our empty_3UTR vector (strong promoter, no UTR) provides a very high level of luciferase signal and serves as a positive control for transfection efficiency, and a synthetic miRNA target reporter vector may serve as a positive control for mimic activity. In addition, we recommend using the GoClone 3'UTR housekeeping controls and random controls for accurately separating sequence-specific vs. non-specific effects.
Study your protocol and whenever possible make master mixes to decrease the variability in your data due to pipetting error or evaporation.
This protocol uses DharmaFect Duo transfection reagent (Dharmacon).
Day 1
IMPORTANT TIP: Choose your cell line wisely. For best results, use a cell line that is known to be transfectable. We have also found that the measurable response of a 3' UTR reporter to a miRNA mimic is attenuated in cell lines with high endogenous levels of that particular miRNA. If you don't know how much miRNA your cell line expresses, try SGG's synthetic microRNA target which indirectly measures the abundance of a particular miRNA in a cell as well as informing you as to the dynamic range of your assay.
1. Seed cells to yield &ge;80% confluence at time of transfection.
<ol>
<li>Seed cells in a 96-well white TC plate in 100&micro;l total volume.</li>
<li>In parallel, seed the appropriate number of cells in a clear 96-well plate for assessing confluence.</li>
</ol>
IMPORTANT TIP 1: Confluence is key to achieving good results in a mimic experiment. For optimal knockdown, cells should be 100% confluent at time of transfection.
IMPORTANT TIP 2: Use low passage cells for best results. Cells that have been passaged too many times tend to yield noisy transfection data.
Day 2
Prepare GoClonereporter constructs and miRNAs, then transfect into the confluent cells.
2. Prepare Constructs
<ol>
<li>Thaw GoClone constructs (plasmid DNA) at room temperature. Mix well.</li>
<li>Centrifuge to remove condensation from the cap.</li>
</ol>
3. Prepare Micro RNAs
<ol>
<li>Thaw microRNAs (specific mimics and non-targeting controls) at room temperature and centrifuge to remove condensation from the caps.</li>
<li>Dilute to a working concentration of 2 &micro;M in RNase-free water.</li>
</ol>
In addition to an experimental miRNA mimic ALWAYS include a non-targeting miRNA control. Co-transfecting a plasmid and a small RNA oligo leads to lower signal than transfecting the plasmid alone.
Include one or more positive controls. Consider using one of the hundreds of synthetic miRNA target reporter vectors in SGG's catalog.
Include one or more negative or non-specific controls. SGG has a range of controls including 3' UTRs of housekeeping genes, random fragments and/or the empty (no UTR insert) control. The use of one or more of these controls alongside an experimental construct will allow you to normalize for any non-specific effects attributable to the overexpression of your specific miRNA or the non-targeting control.
4. Prepare Transfections
<ol>
<li>For each transfection combine the following reagents for a single assay:</li>
</ol>
Mixture 1
<table border="1">
<tbody>
<tr><td>Individual GoClone reporter:</td><td>3.33 &micro;L</td></tr>
<tr><td>microRNA:</td><td>Varies*</td></tr>
<tr><td>Serum-free media:</td><td>to 10 &micro;L</td></tr>
</tbody>
</table>
* Effective concentration of miRNA can vary from 10 nM-100 nM. Consult miRNA manufacturer for appropriate concentration.
IMPORTANT TIP: Do not add too much mimic. A reasonable range for mimic concentrations is 10-50 nM in the final 100uL reaction.
Perform at least three replicate transfections for each treatment/UTR combination and include additional volume to allow for pipetting error.
For example, multiply the single reaction volumes by 3.5 to get enough transfection mix for 3 replicate transfection wells including extra for pipetting error or evaporation.
For each reporter, set up replicates for the following: reporter only, reporter + non-targeting control miRNA and reporter + specific miRNA.
Set up transfections for the targeting microRNA at the same time as those for a non-targeting control.
<ol start="2">
<li>Make up the DharmaFECT DUO (Dharmacon) mixture as follows for each transfection:</li>
</ol>
Mixture 2
<table border="1">
<tbody>
<tr><td>DharmaFect Duo:</td><td>0.15 &micro;L</td></tr>
<tr><td>Serum free media:</td><td>9.85 &micro;L</td></tr>
</tbody>
</table>
The amount of DharmaFECT Duo to be added may be cell line dependent.
The amounts indicated above are appropriate for HT1080 cells.
Consult the manufacturer's documentation to determine the appropriate amount for your cell line.
Tip: Make a large transfection mixture by multiplying the volumes above by the number of reporters, the number of replicates and the number of miRNAs to be tested. When calculating the amount of transfection mix to prepare, be sure to include a small amount extra to account for pipetting errors and evaporation.
<ol start="3">
<li>Allow the DharmaFECT Duo mixture to incubate at room temperature for 5 minutes.</li>
<li>After 5 minutes, add 10&micro;L of the DharmaFECT Duo mixture (Mixture 2) to each prepared tube of 10uL plasmid and/or miRNA (Mixture 1). Each tube should then contain a volume of 20&micro;L per replicate transfection.</li>
<li>Incubate each mixture for 20 minutes at room temperature.</li>
<li>After 20 minutes, add 80 &micro;L of pre-warmed (37&deg;C), antibiotic-free, complete media per replicate to each tube for a total of 100&micro;L per replicate transfection. A deep-well block may be used for preparation of many replicates and samples simultaneously. Mix the solution by pipetting up and down.</li>
<li>Remove the seeded plate from the incubator. Verify that cells are at least 80% confluent.</li>
<li>Carefully pipet off the media from each well.</li>
<li>Add 100&micro;L of the transfection mixture (20uL DNA/reagent mix + 80uL media) to each well.</li>
<li>Place plate in incubator overnight.</li>
</ol>
Don't over-incubate. Most mimics will produce significant results on bona fide targets within 24 hours. The level of repression measurable at 48 hours may be greater, but so will the well to well variability.
Mimic experiments are easier than inhibitor experiments. While most mimics will significantly repress true targets within 24 hours, significant de-repression attributable to the inhibitor can take 48 hours to be measurable and will almost always be less pronounced than the effect of the mimic.
Day 3
Note: Luciferase assays may be conducted immediately or the plates may be frozen at -80&deg;C (freezing generally increases cell lysis and luciferase signal). Freeze and thaw your cells before assaying for best results.
REMEMBER to let frozen cells thaw to room temperature before adding LightSwitch Assay Reagent.
5. Measure luciferase activity with LightSwitch AssayReagents
<ol>
<li>Remove plate from incubator and bring to room temperature.</li>
<li>Prepare LightSwitch Assay Reagents (for LS010 kit, protocols of other kit sizes may be found online):
<ol>
<li>Reconstitute 100X Substrate by adding 100&micro;L of Substrate Solvent to tube of lyophilized Assay Substrate. Protect from light and minimize time at room temperature. 100X Substrate may be stored at -20C for 2-3weeks. For best results, use freshly reconstituted substrate.</li>
<li>Prepare Assay Solution by thawing 10mL bottle of Assay Buffer in room temperature water bath and add 100&micro;L of reconstituted 100X Substrate prior to use. Mix well.</li>
</ol>
</li>
<li>Use a multi-channel pipettor to add 100&micro;L LightSwitch Assay Solution (buffer+substrate) directly to each sample well (containing 100uL cells + media) in a white 96-well plate. If cells were grown in another plate or flask format, transfer samples to a white 96-well plate in 100&micro;L total volume (media or PBS).</li>
<li>Cover plate, protect from light, and incubate for 30 minutes at room temperature. If assaying more than one plate, stagger addition of assay solution so that each plate incubates for 30 minutes before reading.</li>
<li>Read each well for 2 seconds in a plate luminometer (SpectraMax L or equivalent).</li>
<li>Calculate the knockdown by calculating luciferase signal ratio for each construct for specific miRNA over the non-targeting control (luminescence = specific miRNA/non-targeting control). Use the data from housekeeping, random, and empty constructs to control for non-UTR specific treatment effects.</li>
</ol>
Representative Results
3'UTR targets in the presence of a targeting miRNA show luciferase knockdown.
The knockdown of 3'UTR-luciferase reporter activity by let-7a was measured in HT1080 cells by co-transfecting 100ng of each reporter construct with 20nM let-7a mimic or non-targeting control, incubating for 24 hours, then reading the luminescent reporter signal using LightSwitch Luciferase Assay Reagents.
<img src="/files/ftp_upload/3343/3343fig1.jpg" alt="Figure 1" /> 
Figure 1. PUNC and ARID3B human 3'UTRs are targets of the let-7a microRNA. Signals from human 3'UTR reporters for the PUNC and ARID3B genes are significantly knocked down in the presence of the let-7a microRNA mimic while signals for housekeeping and random sequence UTRs are not.

<ol>
	<li>Griffiths-Jones, S., Grocock, R. J., van Dongen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+microRNA+sequences,+targets+and+gene+nomenclature.">miRBase: microRNA sequences, targets and gene nomenclature.</a> Nucleic Acids Res. 34, D140-D144 (2006).</li><li>Calin, G. A., Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+cancer+connection:+the+beginning+of+a+new+tale.">MicroRNA cancer connection: the beginning of a new tale.</a> Cancer Res. 66, 7390-7394 (2006).</li><li>Chen, J. F., Murchison, E. P., Tang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+deletion+of+Dicer+in+the+heart+leads+to+dilated+cardiomyopathy+and+heart+failure.">Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 6-6 (2008).</li><li>Baek, D., Villen, J., Shin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+microRNAs+on+protein+output.">The impact of microRNAs on protein output.</a> Nature. 455, 64-71 (2008).</li><li>Hendrickson, D. G., Hogan, D. J., Herschlag, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+identification+of+mRNAs+recruited+to+argonaute+2+by+specific+microRNAs+and+corresponding+changes+in+transcript+abundance.">Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance.</a> PLoS One. 3, e2126-e2126 (2008).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2009).</li><li>Lall, S., Krek, A., Chen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+map+of+conserved+microRNA+targets+in.">A genome-wide map of conserved microRNA targets in.</a> 16, 460-471 (2006).</li><li>Boutz, D. R., Collins, P. J., Suresh, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-tiered+approach+identifies+a+network+of+cancer+and+liver+disease-related+genes+regulated+by+miR-122.">Two-tiered approach identifies a network of cancer and liver disease-related genes regulated by miR-122.</a> J. Biol. Chem. 286, 18066-18078 (2011).</li><li>Lim, L. P., Lau, N. C., Garrett-Engele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+shows+that+some+microRNAs+downregulate+large+numbers+of+target+mRNAs.">Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.</a> Nature. 433, 769-773 (2005).</li><li>Selbach, M., Schwanhausser, B., Thierfeldner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+changes+in+protein+synthesis+induced+by+microRNAs.">Widespread changes in protein synthesis induced by microRNAs.</a> Nature. 455, 58-63 (2008).</li><li>Hutvagner, G., Simard, M. J., Mello, C. C., Zamore, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+inhibition+of+small+RNA+function.">Sequence-specific inhibition of small RNA function.</a> PLoS Biol. 2, e98-e98 (2004).</li><li>Meister, G., Landthaler, M., Dorsett, Y., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+inhibition+of+microRNA-+and+siRNA-induced+RNA+silencing.">Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing.</a> RNA. 10, 544-5450 (2004).</li><li>Liao, J. M., Lu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auto-regulatory+suppression+of+c-Myc+by+miR-185-3p.">Auto-regulatory suppression of c-Myc by miR-185-3p.</a> J. Biol. Chem. Epub. , (2011).</li></ol>

The authors are employees of SwitchGear Genomics who make tools and reagents appearing in this article. Production of this article was sponsored by SwitchGear Genomics.

We have created a genome-wide collection of pre-cloned human 3'UTR-reporter constructs to enable studies of miRNA-3'UTR interactions in living cells. Here we have shown a reproducible and high-throughput method for co-transfecting miRNA mimics with 3'UTR-reporters using a luciferase reporter system.
This method relies on researchers using a highly-transfectable cell line and perturbing miRNA activity with an overexpression technique. Researchers using poorly-transfectable cell lines may consider using our companion lentiviral 3'UTR-reporter collection created in conjunction with Sigma (Mission 3'UTR Lenti GoClones). Treating the cells with miRNA mimics results in an overexpression-type experiment, and with any such study, researchers must take into account that this may or may not represent actual physiological conditions. To address overexpression concerns, researchers may use miRNA inhibitors in addition to mimics to study the interaction with two different types of perturbation12-14.
Once likely miRNA targets are identified, researchers may wish to mutagenize the putative miRNA binding sites to determine whether the sites are necessary for the functional interaction. They may also wish to scale up to testing hundreds of different potential target UTRs (Boutz). In addition, if researchers are interested as to whether the miRNA is affecting transcript stability or translation efficiency, luciferase reporter data may be compared to measures of steady-state transcript levels. Any change in luciferase signal that is not associated with a proportional change in transcript levels will point to an effect on translation efficiency. Finally, researchers may use reporter vectors create a dose response curve to compare the relative sensitivity of different UTRs to changes in miRNA concentration14.

<table border="1">
<tbody>
<tr><td>&nbsp;</td><td>Vendor</td><td>Catalog Number</td></tr>
<tr><td>White Tissue Culture Plates (96-well solid bottom)</td><td>VWR</td><td> 82050-736</td></tr>
<tr><td>Clear Tissue Culture Plates (96-well) with lid</td><td>VWR</td><td>353072</td></tr>
<tr><td><a href="http://switchgeargenomics.com/jove/">LightSwitch Luciferase Assay Reagent</a></td><td><a href="http://switchgeargenomics.com/jove/">SwitchGear</a></td><td><a href="http://switchgeargenomics.com/jove/">LS010</a></td></tr>
<tr><td>DharmaFECT Duo Transfection Reagent (0.75mL)</td><td>Dharmacon</td><td>T-2010-02</td></tr>
<tr><td>Foil Plate Sealing Tape</td><td>E&amp;K Scientific</td><td>T592100</td></tr>
<tr><td>Breathable Plate Sealing Tape</td><td>E&amp;K Scientific</td><td>T896100-S</td></tr>
<tr><td>Plate Luminometer</td><td>Molecular Devices</td><td>SpectraMax L</td></tr>
</tbody>
</table>

identifying targets of human micrornas with the lightswitch luciferase assay system using 3'utr-reporter constructs and a microrna mimic in adherent cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Watch this Scientific Journal Video about Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells at 

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

identifying-targets-human-micrornas-with-lightswitch-luciferase-assay

Research

JoVE Journal

Biology

36.1K Aufrufe.  SwitchGear Genomics. MicroRNAs (miRNAs) sind wichtige Regulatoren der Genexpression und haben gezeigt, dass eine Rolle bei zahlreichen biologischen Prozessen spielen. Um besser zu verstehen miRNA-UTR-Interaktionen, haben wir eine genomweite Sammlung von 3 UTR Luciferase Reporter mit einem neuartigen Luciferase-Gen und Assay-Reagenz, das LightSwitch System gepaart erstellt.

Serie: Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

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.

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

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.