Name: High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes (Video) | JoVE Journal
Uploaded: 2015-03-03T15:00:00.000+00:00
Duration: 9 min 44 s
Description: Learn a cell-based reporter gene assay to screen chemical libraries for modulators of post-transcriptional gene regulation via the 3' UTR. This method enables high-throughput identification of compounds affecting mRNA stability.

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5 minutes

We thank the Italian Neuroblastoma Foundation for the full financial support to this project.

NOTE: The throughput of such assay systems depends on the available HTS lab equipment. This protocol is facilitated by a Tecan Freedom EVO 200 robot, which performs liquid handling in 96-well format. Miniaturization to 384-well format is also possible. The robotic liquid handling system is positioned under a laminar flow hood in order to maintain aseptic conditions during all experimental steps. If no liquid-handling automation is available, the protocol can be readily adapted to low-throughput format.
Primary Screening
1. Day 1: Prepare and Seed Cells
NOTE: Seed cells to yield 80% confluence at the time of assaying luciferase activity, i.e., 48 hr after seeding.
<ol>
	<li>Resuspend trypsinized CHP134-mycn3UTR neuroblastoma cells to a concentration of 2 &#215; 105 cells/ml in RPMI containing 10% FBS and 1% L-glutamine. For 27 plates prepare at least 250 ml of cell suspension taking into account liquid handling, troughs dead volumes and repeated pipetting steps. Pour the cell suspension into sterile reservoir and place it on the robot desk.</li>
	<li>Place 9 barcoded white flat-bottom 96-well plates and a box of 200 &#956;l sterile pipet tips on the robot desk. Mix the cell suspension by pipetting before each dispensation step to avoid cells settling under gravity. Dispense 75 &#956;l of cells into each well of 9 plates to get a final density of 1.5 &#215; 104 cells per well.</li>
	<li>Cover the plates with the lids and leave them outside of the incubator for 30 min to allow the cells to sediment to the bottom of the well. This will minimize edge effects.</li>
	<li>Repeat steps 1.2 and 1.3 twice in order to prepare a total number of 27 plates.</li>
	<li>Place the plates at 37 &#176;C and 5% CO2 and incubate for 24 hr.</li>
</ol>
2. Day 2: Treat the Cells with Library Compounds
NOTE: The Spectrum Collection small molecule library consists of 2,000 compounds arranged in 8 rows and 10 columns in 25 96-well plates at the concentration of 10 mM in DMSO. The wells on the first and last columns are left empty for controls. Compound screening assays are typically performed at 1&#8211;10 &#181;M compound concentration8. The screening protocol described here fixes 2 &#956;M as the working concentration and 24 hr as the assay end point.
<ol>
	<li>Thaw 9 compound library plates at room temperature.</li>
	<li>Prepare two troughs with sterile PBS and PBS-0.5% DMSO solutions and place them on the robot desk. For each library plate, prepare and label accordingly one dilution plate &#8212; a polypropylene U-bottom 96-well plate with a working volume of at least 400 &#956;l. Fill each well of columns 2 to 11 of dilution plates with 398 &#956;l of sterile PBS using the liquid handler&#8217;s fixed tips. Fill columns 1 and 12 with 50 &#956;l of sterile PBS with 0.5% DMSO in order to reproduce the vehicle concentration in the control wells (0.02%).</li>
	<li>Place two library plates, the correspondingly labeled dilution plates, two boxes of 50 &#956;l sterile pipet tips and six cells plates on the robot desk. Uncover cells plates.</li>
	<li>Pipet 2 &#956;l of the 10 mM stock solution from one of two library plates in the corresponding dilution plate and mix well. This will result in an intermediate compounds concentration of 50 &#956;M. Using the same set of tips, dispense 3 &#956;l of the 50 &#956;M dilution in 3 barcoded white 96-well plates with cells. This dilution scheme results in 2 &#956;M working concentration of each tested compound. 
	NOTE: To avoid unnecessary manipulations with the tips, use a full set of 96 tips from the beginning, even though the library plates contain only 80 compounds. This is possible since the first and the last columns of the library plates are empty.</li>
	<li>Replace the pipet tips and repeat Step 2.4 for the second library plate.</li>
	<li>Cover the cells plates and return them to the incubator for 24 hr.</li>
	<li>Repeat Steps 2.3-2.6 two more times for the remaining library plates.</li>
</ol>
3. Day 3: Perform Luciferase Assay
NOTE: Before performing the luciferase assay, it is possible to multiplex it with a cell viability assay based on reduction of resazurin in order to obtain a cell-viability index from each well9. Multiplexing luciferase and viability assay assays results in a reduction of luciferase signal that, however, can be ignored if the cells provide high enough level of luciferase activity. Luciferase activity can be detected by a variety of commercial and homemade10,11 luciferase assay reagents with stable luminescent signal.
<ol>
	<li>Equilibrate the reconstituted luciferase reagent of choice to room temperature. Make sure the volume is enough for all the assayed plates. For 27 plates 170 ml of luciferase reagent will be required taking into account liquid handling, troughs dead volume and repeated pipetting steps. Pour the luciferase reagent into a reservoir and place it on the robot desk.</li>
	<li>Remove 9 plates with cells from the incubator, place them on the robot desk, uncover and wait until equilibrated to room temperature. Add 50&#160;&#956;l of the luciferase reagent per well plate by plate. Between the plates, introduce a delay equal to the luminescence reading time of one plate. This will assure that all the plates are measured within equal time interval from the reagent addition.</li>
	<li>After a 30 min incubation, place the first plate in the luminometer and measure luminescence after a brief shaking step. Repeat for all plates. Record the barcode of each plate and associate it to the corresponding data file to avoid mistakes arising from a large number of handled plates.</li>
	<li>Repeat Steps 3.2-3.3 twice for the remaining 18 plates with cells.</li>
	<li>Collect the remaining luciferase reagent and store it at -20 &#176;C.</li>
</ol>
4. Day 4: Data Analysis
<ol>
	<li>Process experimental data using normalization of all samples to the average of on-plate vehicle-treated controls. For each library compound, calculate the mean value x&#773; and SD over triplicates.</li>
	<li>Select up-regulating and down-regulating hits by setting a threshold of x&#773; &#177; k &#215; SD, where the mean value x&#773; and SD are computed over all assay processed values, and k is a defined constant.</li>
	<li>Select an arbitrary threshold cut-off &#60; 20% of vehicle-treated controls when a reduction of luciferase signal is most probably associated with compound toxicity. Discarding the hits below this threshold will significantly reduce a number of false-positive hits in the counter-screening. 
	NOTE: Instead of control-based normalization, experimental data can be processed applying Z score or its robust analog B score12. In addition, alternative statistical approaches for hit selection are available12.</li>
</ol>
5. Counter-screening
NOTE: Compounds identified in the primary screen (labeled &#8216;hits&#8217;) are confirmed and evaluated for the specificity by a counter-screening.
<ol>
	<li>Cherry pick the selected hits from the aliquots stored in single 2D barcoded tubes and create new &#8220;hit plates&#8221;, saving the corresponding layout. Do not forget to leave free positions for controls. 
	NOTE: In the absence of an efficient cherry-picking system, one might test both the control and 3&#8217; UTR-bearing cells in parallel in the primary screening. In this case, specific selection of compounds with effects dependent only on the introduced 3&#8217; UTR will be possible after the primary screening eliminating the need of counter-screening. However, the parallel screening in two cell lines will double the assay costs.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 1: Prepare and Seed Cells&#8221;), for each &#8220;hit plate&#8221; prepare three 96-well plates of each CHP134-mycn3UTR and CHP134-CTRL stable cells.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 2: Treat the Cells with Library Compounds&#8221;), use each hit plate to treat three CHP134-mycn3UTR and three CHP134-CTRL plates at 2 &#956;M working concentration. 
	NOTE: While the counter-screen described here is performed in triplicates at the single dose of 2 &#956;M, it might be beneficial to substitute the standard replicates and test the hits in 3 concentrations as 10-fold dilutions ranging from 2 &#956;M to 20 nM. Applying this approach would not only allow to confirm the data of the primary screen, but also to obtain preliminary dose-response data on the primary hits, minimizing false-positive rates. In this case, a different analysis pipeline should be applied to process the experimental data and confirm the hits.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 3: Perform Luciferase Assay&#8221;), measure luciferase activity in all CHP134-mycn3UTR and CHP134-CTRL plates.</li>
	<li>Following the protocol for the primary screening (section &#8220;Day 4: Data Analysis&#8221;), normalize experimental data from treated wells to the average of on-plate vehicle-treated controls and calculate the mean and SD over replicates. For each compound tested, compute the fold change of luciferase activity in CHP134-mycn3UTR vs. CHP134-CTRL cells. Select arbitrary fold change cut-offs of &#62;1.5 and significance p-values of &#60;0.01. 
	NOTE: In the described assay settings the determinant parameter for hit specificity is a significant fold change of luciferase activity in CHP134-mycn3UTR vs. CHP134-CTRL cells. If needed, one might also assess compound toxicity by performing concurrently a cell viability assay on the selected compounds. This might be especially beneficial if the primary hits are counter-screened in a dose response analysis. For a single dose, our experience indicates a strong correlation of reduced luciferase activity with decreased cell viability6. Therefore, decreased luciferase activity in target cells as well as control cells can be considered as an indicator of compound toxicity at the tested concentration.</li>
</ol>

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<li>Cheung, N. -K. V., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroblastoma%3A+developmental+biology%2C+cancer+genomics+and+immunotherapy%5BTitle%5D)+AND+%22Nat.+Rev.+Cancer%22%5BJournal%5D)">Neuroblastoma: developmental biology, cancer genomics and immunotherapy.</a> Nat. Rev. Cancer. 13 (6), 397-411 (2013).</li>
<li>Hughes, J. P., Rees, S., Kalindjian, S. B., Philpott, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+of+early+drug+discovery%5BTitle%5D)+AND+%22Br.+J.+Pharmacol%22%5BJournal%5D)">Principles of early drug discovery.</a> Br. J. Pharmacol. 162 (6), 1239-1249 (2011).</li>
<li>Herbst, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplexing+a+high-throughput+liability+assay+to+leverage+efficiencies%5BTitle%5D)+AND+%22Assay+Drug+Dev.+Techn%22%5BJournal%5D)">Multiplexing a high-throughput liability assay to leverage efficiencies.</a> Assay Drug Dev. Techn. 7 (3), 294-303 (2009).</li>
<li>van Olst, E. S. iebring-, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Affordable+luciferase+reporter+assay+for+cell-based+high-throughput+screening%5BTitle%5D)+AND+%22J.+Biomol.+Screen%22%5BJournal%5D)">Affordable luciferase reporter assay for cell-based high-throughput screening.</a> J. Biomol. Screen. 18 (4), 453-461 (2013).</li>
<li>Baker, J. M., Boyce, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+functional+screening+using+a+homemade+dual-glow+luciferase+assay%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">High-throughput functional screening using a homemade dual-glow luciferase assay.</a> J. Vis. Exp. (88), (2014).</li>
<li>Malo, N., Hanley, J. A., Cerquozzi, S., Pelletier, J., Nadon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Statistical+practice+in+high-throughput+screening+data+analysis%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">Statistical practice in high-throughput screening data analysis.</a> Nat. Biotechnol. 24 (2), 167-175 (2006).</li>
<li>Hitti, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+versatile+ribosomal+protein+promoter-based+reporter+system+for+selective+assessment+of+RNA+stability+and+post-transcriptional+control%5BTitle%5D)+AND+%22RNA%22%5BJournal%5D)">A versatile ribosomal protein promoter-based reporter system for selective assessment of RNA stability and post-transcriptional control.</a> RNA. 16 (6), 1245-1255 (2010).</li>
<li>Oikonomou, P., Goodarzi, H., Tavazoie, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systematic+identification+of+regulatory+elements+in+conserved+3%E2%80%99+UTRs+of+human+transcripts%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Systematic identification of regulatory elements in conserved 3’ UTRs of human transcripts.</a> Cell Rep. 7 (1), 281-292 (2014).</li>
<li>Gandin, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polysome+fractionation+and+analysis+of+mammalian+translatomes+on+a+genome-wide+scale%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.</a> J. Vis. Exp. (87), (2014).</li>
<li>Conne, B., Stutz, A., Vassalli, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+3%E2%80%99+untranslated+region+of+messenger+RNA%3A+A+molecular+%E2%80%9Chotspot%E2%80%9D+for+pathology%5BTitle%5D)+AND+%22Nat.+Med%22%5BJournal%5D)">The 3’ untranslated region of messenger RNA: A molecular “hotspot” for pathology.</a> Nat. Med. 6 (6), 637-641 (2000).</li>
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<li>Pascale, A., Govoni, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+complex+world+of+post-transcriptional+mechanisms%3A+is+their+deregulation+a+common+link+for+diseases%3F+Focus+on+ELAV-like+RNA-binding+proteins%5BTitle%5D)+AND+%22Cell.+Mol.+Life+Sci%22%5BJournal%5D)">The complex world of post-transcriptional mechanisms: is their deregulation a common link for diseases? Focus on ELAV-like RNA-binding proteins.</a> Cell. Mol. Life Sci. 69 (4), 501-517 (2012).</li>
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</ol>

The authors declare that they have no competing financial interests.

This protocol describes a cell-based reporter-gene assay aiming at the identification of modulators that target 3&#8217; UTR-dependent post-transcriptional processes. It encompasses the primary screening and the counter-screen and, if needed, can be accompanied by a cytotoxicity assay. The outcome of the primary screening is a number of valuable candidate compounds, whose reproducibility and specificity is further validated in the counter-screening.
The identification of primary hits is based ...

For a long period transcriptional regulation of gene expression was thought to play a major if not exclusive role in controlling protein production. Accumulating evidence, however, indicates that post-transcriptional regulation contributes as much as, if not more than, transcriptional regulation to determine cellular protein abundance1,2. Post-transcriptional control of gene expression is much more complex and elaborate than was at first thought. In fact, all the various stages of post-transcriptional control have emerged to be regulated, including mRNA processing, localization, turnover, translation3 as well as the newly described reversible RNA methylation4. From a number of post-transcriptional regulation processes potentially affected, the assay described below focuses on those involving the mRNA 3&#39; untranslated region (3&#8217; UTR). 3&#8217; UTR broadly affects mRNA fate mainly via specific interaction of RNA-binding proteins and non-coding RNAs to its regulatory sequences and/or secondary structures5. Therefore, small molecules that alter these interactions or interfere with upstream signal transduction pathways will shift the balance that regulates protein abundance. This therapeutic approach is of particular importance for human pathologies for which evidence exists showing that disease-relevant genes are subjected to post-transcriptional regulation, which thus represents a potential target for pharmacological intervention. Therefore, systems allowing for screening of mRNA levels&#8217; modulators through interference with post-transcriptional control mechanisms in a high-throughput format can become valuable tools in the identification of potential novel treatments.
The described cell-based reporter gene assay allows for the identification of compounds able to modulate mRNA fate via mechanisms dependent on its 3&#8217; UTR. It consists of several principal components (Figure 1) and requires few preliminary steps to validate the feasibility of the assay. First of all, the reporter construct is designed to include the 3&#8217; UTR from a gene of interest. The 3&#8217; UTR is fused to the end of a reporter gene such as firefly luciferase (Figure 1). Any changes in post-transcriptional control mechanisms exerted through the inserted 3&#8217; UTR will alter the stability and/or translation efficiency of this chimeric transcript, resulting in varied luciferase levels. Therefore, changes in luciferase activity serve as indirect measure of affected post-transcriptional regulation of the gene of interest. The second component of the system is a control reporter construct, an identical expression plasmid that expresses the same reporter gene but does not contain any 3&#8217; UTR. The two reporter plasmids can be designed in the lab using conventional molecular biology protocols or purchased from commercial sources.
The selection of an appropriate cell line is critical for the assay construction. Which cell line is the optimal model depends on numerous factors ranging from clinical requirements like maximal resemblance to pathology to just practical issues like availability, transfectability, and growth characteristics. Importantly, prior to proceed one should make sure that fusion of 3&#8217; UTR to the reporter gene has an expected effect on the level of the chimeric transcript. The absence of significant difference in luciferase signal from the 3&#8217; UTR-bearing and control reporter constructs transiently transfected in the selected cells would indicate that the 3&#8217; UTR is not functional in this cellular model, prompting for search of alternative ones. For the high-throughput format, the 3&#8217; UTR-bearing and control luciferase reporter constructs should be stably integrated in the selected cell line. Using a stably integrated over transiently transfected cell line is preferable for several reasons. This will broaden the choice of a cellular model including hard-to-transfect cell lines, decrease the variability in the data arising from fluctuations in transfection efficiency, subside the costs. Moreover, upon transient transfection cells are very often overloaded with a DNA plasmid leading to the saturation of the system. In these settings a further increase in the reporter expression might be challenging, resulting in decreased sensitivity towards potential upregulating compounds.
Finally, when all the assay components are set up, it is critical before moving to the high-throughput format to determine the feasibility of the assay, i.e., if luciferase activity can be indeed up- and down-regulated specifically via the inserted 3&#8217; UTR. The best controls would be small molecules reported to modulate the stability or translatability of mRNA through the 3&#8217; UTR of interest. If having these is not possible, surrogate controls imitating the desired effect are accepted. These could be miRNAs or RNA-binding proteins known to exert their effects via binding to the 3&#8217; UTR of interest. Evaluation of such controls before the primary screening not only indicates the functionality of the developed assay, but also allows for estimation of its dynamic range, sensitivity and performance in the high-throughput format.
Using the described protocol we screened a 2,000-compound library6. Neuroblastoma, the most common extracranial solid tumor of infancy, was used as a biological model and the 3&#8217; UTR of the MYCN oncogene, whose amplification strongly predicts adverse outcome of neuroblastoma, as a target gene7. Figure 2 outlines the experimental layout. The screening was divided in three runs with the batch size of 27 plates screened in one run. The batch of 27 plates included Spectrum Collection library plates n.1-n.8+n.25 (first run), n.9-n.16+n.25 (second run) and n.16-n.24+n.25 (third run), each plate screened in triplicate. Thus, one library plate (n.25) was assayed within all three runs making possible inter-run comparison. The protocol below describes a single run of 9 library plates tested in triplicate.

<table><tbody><tr><td>Culture plate 96-well White</td><td>PerkinElmer</td><td>6005688</td><td></td></tr><tr><td>StorPlate 96-well U bottom (dilution plate)</td><td>PerkinElmer</td><td>6008190</td><td></td></tr><tr><td>100 ml disposable trough for reagents</td><td>Tecan</td><td>10 613 048</td><td></td></tr><tr><td>300 ml disposable robotic reservoir</td><td>VWR</td><td>PB12001301</td><td></td></tr><tr><td>Robot tips DITI 50&amp;nbsp;&amp;mu;l Sterile</td><td>Tecan</td><td>30038607</td><td></td></tr><tr><td>Robot tips DITI 200&amp;nbsp;&amp;mu;l Sterile</td><td>Tecan</td><td>30038617</td><td></td></tr><tr><td>Matrix 1.4 ml 2D Barcoded w, Flat Bottom Tubes</td><td>Thermo Scientific</td><td>3711</td><td></td></tr><tr><td>ONE-Glo Luciferase Assay System&amp;nbsp;</td><td>Promega</td><td>E6120</td><td></td></tr><tr><td>RPMI</td><td>Lonza</td><td>BE12-918F</td><td></td></tr><tr><td>PBS-1x, w/o Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Lonza</td><td>BE17-516F</td><td></td></tr><tr><td>L-Glutamine 200&amp;nbsp;mM</td><td>Lonza</td><td>BE17-605E</td><td></td></tr><tr><td>FBS</td><td>Lonza</td><td>DE14-801F</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>The Spectrum collection (compound library)</td><td>MicroSource Discovery Systems</td><td>N/A</td><td></td></tr><tr><td>Tecan Freedom EVO200 robot&amp;nbsp;</td><td>Tecan</td><td>N/A</td><td></td></tr><tr><td>Tecan F200 multiplate reader&amp;nbsp;</td><td>Tecan</td><td>N/A</td><td></td></tr></tbody></table>

high-throughput screening for chemical modulators of post-transcriptionally regulated genes

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Using the described approach, we screened a 2,000-compound library for potential modulators of post-transcriptional control mechanisms exerted through the 3’ UTR of the MYCN gene. Figure 3 depicts the results of the primary screening exemplified by a single library plate. Luciferase signal displayed as percentage of vehicle-treated controls was obtained by measuring luciferase activity in triplicate plates of CHP134-mycn3UTR cells treated with compounds of a single library plate. As expected, th...

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

This experiment screens chemical libraries for compounds that modulate post transcriptional control mechanisms via the three prime untranslated region of transcripts. First, identify the three prime untranslated region of interest in the targeted gene. Clone this three prime UTR into El luciferase control reporter plasmid to create El luciferase three prime UTR chimeric transcript.Then stably. Integrate the two plasmids into a disease relevant cell line seed the cells carrying the chimeric transcript in 96. Well plates treat them with a library of compounds for 24 hours and then identify the molecules that affect luciferase activity.Cherry pick the selected molecules for further validation screening, and next, treat both the control cells and the cells carrying luciferase three prime UTR chimeric transcript with a cherry picked library subset. Results will differentiate if the effect of a compound is mediated via the three prime untranslated region or via other vector sequences. Unlike the most reporter based screening essays.Assays that focus on transcription regulation, these report cell based assay focus on identification of compounds that modulate mRNA transcript levels via three prime and translated region. Harvest the stably transfected neuroblastoma cells by trypsin Asian. Dilute the cells to a concentration of two times 10 to the fifth cells per milliliter.In RPMI containing 10%FBS and 1%L-glutamine for 27 plates. Prepare at least 250 milliliters of cell suspension, taking into account liquid handling, trough dead volumes, and repeated pipetting steps. Then pour the cell suspension into a sterile reservoir and place it on the robot desk.Load nine barcoded white flat. Bottom 96 well plates and a box of 200 microliter sterile pipette tips on the robot desk. Mix the cell suspension, then dispense 75 microliters of cells into each well of nine plates.Cover the plates and allow the cells to sediment to the bottom of the well for 30 minutes in order to minimize edge effects. Then place all nine plates at 37 degrees Celsius and 5%carbon dioxide and incubate for 24 hours. Similarly, proceed with the remaining plates nine at a time.Thaw the compound library plates at room temperature. Fill one trough with sterile PBS and another one with 0.5%DMSO in PBS solution and place them on the robot desk. For each library plate.Prepare and label accordingly. One dilution plate. Now fill each well of columns, two to 11 of the dilution plates with 398 microliters of sterile PBS.Then fill columns one and 12 with 50 microliters of 0.5%DMSO in sterile PBS. Next load the two dilution plates. Two library plates, two boxes of 50 microliters sterile tips and six cells plates on the robot desk.Now uncover the cell plates, pipette two microliters of the 10 millimolar stock solution from one of two library plates in the corresponding dilution plate and mix well. Using the same set of tips. Transfer three microliters of the 50 micromolar dilution into the three barcoded white 96 well plates containing the cells.Replace the full set of tips and repeat cell inductions for the second library plate. Cover the cultures and incubate for 24 hours. Then repeat the compound dilution and cell induction process for the remaining library plates.Equilibrate the reconstituted luciferase reagent of choice to room temperature. Place the luciferase reservoir on the robot desk and pour reagent into it. Remove nine plates of induced cultures from the incubator.Place them on the robot desk, uncover and equilibrate to room temperature. Then add 50 microliters of the luciferase reagent per well between the plates. Introduce a delay equal to the luminescence reading time of one plate.After a 30 minute incubation. First record the barcode of the plate in order to associate it with the corresponding data file. Next, place the first plate in the luminometer and measure luminescence.After a brief shaking step, repeat the luminescence readings for all plates of inductions. Then collect the remaining Lucifer race reagent and store it at negative 20 degrees Celsius. Once the experimental data of the primary screening are processed, select the hits to confirm compound specificity by a counter screening cherry.Pick the selected hits and create a layout for the new screening plate, including positions for controls. Then from the aliquots stored in single 2D barcoded tubes, create new HIIT plates. Following the same protocol is for the screening.Prepare 3 96 well plates of each stable cell line per hi plate. The next day, treat the cells with library compounds. Use each hi plate to treat three plates of CHP 1 34 micken, three prime UTR and three plates with control cells at two micromolar working concentration in 24 hours.Measure luciferase activity in all CHP 1 34 plates, the three prime untranslated and control sets. As with the data analysis in the primary screening, normalize experimental data from treated wells to the average of on plate vehicle treated controls. Then calculate the mean and standard deviation over the replicates for each compound tested compute.The fold change of luciferase activity in CHP 1 34 McIn three prime UTR versus CHP 1 34. Control cells select arbitrary full change cutoffs of greater than 1.5 and significance P values of less than 0.01. This experiment screened a 2000 compound library for potential modulators of post transcriptional control mechanisms exerted through the three prime UTR of the mic and oncogene the results of luciferase signals of a single out of 25 representative library plate displayed as a percentage of vehicle treated.Controls show that the majority of compounds elicited no apparent effect on the three prime UTR. The hits were selected where the mean value and standard deviation were computed over all assay values compounds reducing luciferase activity below 20%are most likely indicative of pronounced cellular toxicity of the compound. This graph shows representative results for counter screening of four selected compounds designated WX, Y, and Z.Both compounds W and X result in increased luciferase expression from the oncogene three prime UTR reporter as indicated by fold change difference and T-test Note that while effects of both compounds W and X are truly dependent on three prime UTR compound W has no significant effect on cell viability.While compound X displays some cytotoxic activity as indicated by normalized luciferase signal in CHP 1 34 control cells, neither compound Y nor compound Z at two micromolar concentration display A three prime UTR dependent effect on luciferase levels. The decreased luciferase activity detected in primary screening upon treatment with compound Y is rather a consequence of cytotoxicity. In the case of compound Z, the increased luciferase activity is due to unspecific in terms of three prime UTR activation of viral promoter.Since an equivalent upregulation is observed in both control and untranslated sets In this high throughput assay, remember that validating the primary heat in the control cells is critical for discrimination of the compounds that act truly through the three prime untranslated region from those compounds that operate via promoter.

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Biology

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