Name: qpcrtag analysis - a high throughput, real time pcr assay for sc2.0 genotyping
Uploaded: 2015-05-25T15:00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping at JoVE.com

This work was supported in part by National Science Foundation Grant MCB-0718846 and Defense Advanced Research Projects Agency Contract N66001-12-C-4020 (to J.D.B). L.A.M. was funded by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada. &#160;Publication of this article is sponsored by Roche.

1. Prepare Yeast Genomic DNA (gDNA)
<ol>
	<li>Prepare a stock of Yeast Lysis Buffer9 that contains 50 mM Tris pH 8.0, 100 mM NaCl, 1% SDS (w/v), 2% Triton X-100 (v/v), 1 mM EDTA pH 8.0.</li>
	<li>As starting material use ~2 x 106 cells, which typically corresponds to ~100 &#181;l of saturated culture for lab strains of the BY474110 background. Cultured cells should be collected by centrifugation at 1,000 x g for 3 min at room temperature in a 1.5 ml microfuge tube. Aspirate the supernatant.</li>
	<li>Add 200 &#181;l of Yeast Lysis Buffer and resuspend the cell pellet by pipetting.</li>
	<li>Add ~300 &#181;l of acid washed glass beads such that the level of beads is about 1 mm below the level of the cell slurry.</li>
	<li>In a fume hood add 200 &#181;l of phenol/chloroform/isoamyl alcohol (25:24:1). Cap the microfuge tube and ensure that no beads are stuck between the cap and the tube as this will result in leaks during shaking (step 1.6). Invert 3 times to mix and verify the cap is sealed.</li>
	<li>Shake the tube for 10 min at room temperature using a benchtop mixer such as an Eppendorf 5432.</li>
	<li>Centrifuge at 20,800 x g for 10 min at 4 &#176;C.</li>
	<li>Transfer 75 &#181;l of the aqueous phase to a new microfuge tube containing 1 ml of 100% ethanol. Invert 10 times to mix.</li>
	<li>Pellet the DNA by centrifugation at 20,800 x g for 20 min at 4 &#176;C .</li>
	<li>Aspirate the supernatant without dislodging the DNA pellet. Wash the pellet with 500 &#181;l of 70% ethanol.</li>
	<li>Centrifuge at 20,800 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant without dislodging the DNA pellet and air-dry the pellet with the microfuge cap open until excess ethanol has evaporated, which is usually around 10 min.</li>
	<li>Resuspend the DNA pellet in 75 &#181;l of 10mM Tris pH 7.4 and vortex to mix. Store the sample at -20 &#176;C indefinitely. Measure the concentration of DNA in the sample using a benchtop fluorimeter such as the Qubit, which distinguishes DNA from RNA. Ensure that the final concentration of gDNA is ~1-2 ng/&#181;l.</li>
	<li>Prepare a 1:10 dilution of genomic DNA using water and vortex to mix.</li>
	<li>Aliquot 30 &#181;l of diluted gDNA into the appropriate well of a source plate that is compatible with a nanoscale acoustic liquid dispenser (step 3). If using a Labcyte Echo 550, use a 384 well polypropylene plate (384PP plate). 
	NOTE: When sealed appropriately this plate can be stored for at least one month at -20 &#176;C.</li>
</ol>
2. Prepare and Dispense qPCR Master Mix into a 1,536 Multiwell Plate
<ol>
	<li>Prepare 850 &#181;l of qPCR master mix, such as LightCycler 1536 DNA Green Master, in a 1.5 ml microfuge tube and vortex to mix. Centrifuge the qPCR master mix for 2 min at 20,800 x g at room temperature to remove any bubbles. The components of the qPCR master mix required for a single 1,536 multiwell plate are as follows:
	<ol>
		<li>Combine 170 &#181;l of enzyme master mix (Green Master Tube 1, 5x concentrated), 42.5 &#181;l of SYBR (Green Master Tube 4, 20x concentrated), and 637.5 &#181;l of water.</li>
	</ol>
	</li>
	<li>Dispense 500 nl/well into a 1,536 multiwell plate using a bulk liquid dispenser such as the Art Robbins Cobra. (Note that steps 2.2.1-2.2.4 refer specifically to the Cobra.)
	<ol>
		<li>Create a dispensing program with the following settings: liquid class, water; aspirate volume, 800 &#181;l; dispense volume, 500 nl; wash time, 20 sec. In the dispense settings, verify the dispense speed is set to 49.6 mm/sec, the dispense offset is 0.25 mm, and excess aspirated liquid will be dispensed back into the source well.</li>
		<li>Place the microfuge tube containing the qPCR master mix in well A1 of the microfuge tube holder, which is located in deck position 1. Cut off the cap of the microfuge tube or simply leave it open. Set the &#8216;Aspirate well&#8217; to A1 by editing the Aspirate settings.</li>
		<li>Perform a wash step prior to dispensing qPCR master mix by first de-selecting the aspirate and dispense steps of the program set up in step 2.2.2 and then hitting &#8216;run&#8217;. Once complete, re-select the aspirate and dispense steps prior to running the full program to dispense qPCR master mix. If the Cobra has been sitting idle for less than 10 min the initial wash step, which serves to prime the system, is unnecessary.</li>
		<li>Hit &#8216;run&#8217; to dispense qPCR master mix to each well.</li>
	</ol>
	</li>
	<li>Collect the qPCR master mix at the bottom of each well by brief centrifugation of the 1536 multiwell plate (30 sec using a low-speed PCR plate spinner).</li>
</ol>
3. Dispense Template DNA and Primers into the 1,536 Multiwell Plate
<ol>
	<li>Dispense 5 nl of diluted gDNA (step 1.3) into the desired wells of the 1536 multiwell plate using an acoustic liquid transfer system, such as the Echo 550.
	<ol>
		<li>For the Echo 550, use the Plate Reformat software or alternatively provide a custom Excel spreadsheet to program the transfer. Ensure the selected source well for the gDNA in the program matches the source well into which you have physically dispensed the gDNA (step 1.3). Choose the source plate type &#8216;384PP_AQ_BP2&#8217;, which indicates the liquid in the 384PP plate is a buffer without surfactants.</li>
	</ol>
	</li>
	<li>Dispense 10 nl of primers, pre-mixed forward and reverse to a final concentration of 50 &#181;M each, into the desired wells of the 1536 multiwell plate using an acoustic liquid transfer system, such as the Echo 550.
	<ol>
		<li>For the liquid transfer system, use the Plate Reformat software or alternatively provide a custom Excel spreadsheet to program the transfer. 
		NOTE: Here it is possible to dispense primers such that the layout on the 1,536 multiwell plate matches the chromosomal order of the primers so it is easy to visually inspect the real time PCR results later (step 6).</li>
		<li>Premix the primers in a plate that is compatible with the acoustic liquid dispenser. For the Echo 550, use a Labcyte 384LDV (low dead volume) plate and choose the source plate type &#8216;384LDV_AQ_B&#8217; when programming the Echo to indicate the liquid is a simple buffer with no proteins.</li>
	</ol>
	</li>
</ol>
4. Seal and Centrifuge the 1536 Multiwell Plate
<ol>
	<li>Using a microplate sealer system such as the Plateloc Thermal Microplate Sealer, immediately seal the 1,536 multiwell plate using optically clear seal that is compatible with the heat sealer instrument. Do not touch the surface of the seal to avoid smudge marks.
	<ol>
		<li>If using the Plateloc Thermal Microplate Sealer, use the following settings: seal time 2.0 sec, seal temperature 162 &#176;C, air pressure 82 psi. This instrument takes ~5 min to heat up and so should be turned on in advance.</li>
		<li>If using the Plateloc Thermal Microplate Sealer, an adapter must be used to raise the height of the 1536 multiwell plate on the stage of the instrument to ensure good sealing. 
		NOTE: While it is preferable to purchase a Stage Plate for the Plateloc Thermal Microplate Sealer, an alternative solution is to insert four ~2 mm thick standard washers available at any hardware store in the corners to raise the height of the 1,536 multiwell plate.</li>
	</ol>
	</li>
	<li>Immediately centrifuge the sealed 1,536 multiwell plate at 2,000 x g for 3 min at room temperature to collect all reagents at the bottom of each well.</li>
</ol>
5. Real Time PCR
<ol>
	<li>Program a 1,536 qPCR instrument, such as the LightCycler 1536, with a two-step amplification protocol followed by a melt curve analysis. Using the LightCycler 1,536 software set up a program as follows:
	<ol>
		<li>Pre-incubation: 95 &#176;C, 1 min hold, ramp rate 4.8 &#176;C/sec, no acquisition.</li>
		<li>Two Step Amplification: 30 cycles of [95 &#176;C, no hold, ramp rate 4.8 &#176;C/sec, no acquisition; 64 &#176;C, 30 sec hold, ramp rate 2.5 &#176;C/sec, single acquisition].</li>
		<li>Melt curve: 95 &#176;C, 10 sec, ramp rate 4.8 &#176;C/sec, no acquisition; 60 &#176;C, 1 min, ramp rate 2.5 &#176;C/sec, no acquisition; 97 &#176;C, ramp rate 0.1 &#176;C/sec, 5 acquisitions/&#176;C (continuous).</li>
		<li>Cool: 40 &#176;C, 30 sec, ramp rate 2.5 &#176;C/sec, no acquisition.</li>
	</ol>
	</li>
	<li>Set the Detection Format to &#8216;Green Intercalating Dye&#8217; in the Run Definition tab.</li>
	<li>Set the pipetting control to &#8216;Master Control&#8217; in the Run Definition tab. This feature serves as an internal control and detects the presence of qPCR master mix in each well of the 1,536 multiwell plate, which is useful to identify putative false negatives.</li>
	<li>Insert the 1536 multiwell plate prepared in steps 2-4 into the 1,536 qPCR instrument and run the program.</li>
</ol>
6. Data Analysis
<ol>
	<li>Perform visual inspection of the data within the qPCR software. 
	NOTE: The 1,536 software enables visualization of both amplification and melt curves, as well as a heat map showing the call (positive, negative, invalid, N/A; Figure 2), the crossing point value (sliding color scale for positive or gray for negative; Figure 3), or an endpoint fluorescence (EPF) value (sliding color scale).</li>
	<li>Export data from the 1,536 Software in the form a .txt file (results table) or .xml file with either the raw data or calculated data for processing offline from the instrument. 
	NOTE: The proprietary automated crossing point (Cp) calling algorithm built into the 1,536 software takes into account the shape and slope of the amplification curve while determining positive/negative and Cp value calls with high confidence, at low sub microliter reaction volumes with comparatively low fluorescence values.</li>
	<li>If the LightCycler DNA Green Master was used for qPCR, verify that all wells passed the &#8216;master control&#8217;. A fail indicates the well did not receive qPCR master mix and the missing data point should be considered a potential false negative. Verify that all wells passed the &#8216;master control&#8217;. A fail indicates the well did not receive DNA mastermix and consider the missing data point a potential false negative.</li>
</ol>

<ol>
	<li>Annaluru, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+synthesis+of+a+functional+designer+eukaryotic+chromosome.">Total synthesis of a functional designer eukaryotic chromosome.</a> Science. 344 (6179), 55-58 (2014).</li><li>Dymond, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+chromosome+arms+function+in+yeast+and+generate+phenotypic+diversity+by+design.">Synthetic chromosome arms function in yeast and generate phenotypic diversity by design.</a> Nature. 477 (7365), 471-476 (2011).</li><li>Goffeau, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+with+6000+genes.">Life with 6000 genes.</a> Science. 274 (5287), 546-547 (1996).</li><li>Dymond, J., Boeke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Saccharomyces+cerevisiae+SCRaMbLE+system+and+genome+minimization.">The Saccharomyces cerevisiae SCRaMbLE system and genome minimization.</a> Bioeng Bugs. 3 (3), 168-171 (2012).</li><li>Richardson, S. M., Nunley, P. W., Yarrington, R. M., Boeke, J. D., Bader, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GeneDesign+3.0+is+an+updated+synthetic+biology+toolkit.">GeneDesign 3.0 is an updated synthetic biology toolkit.</a> Nucleic Acids Res. 38 (8), 2603-2606 (2010).</li><li>Richardson, S. M., Liu, S., Boeke, J. D., Bader, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-A-Gene+with+GeneDesign.">Design-A-Gene with GeneDesign.</a> Methods Mol Biol. 852, 235-247 (2012).</li><li>Lajoie, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+limits+of+genetic+recoding+in+essential+genes.">Probing the limits of genetic recoding in essential genes.</a> Science. 342 (6156), 361-363 (2013).</li><li>Jovicevic, D., Blount, B. A., Ellis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+synthesis+of+a+eukaryotic+chromosome:+Redesigning+and+SCRaMbLE-ing+yeast.">Total synthesis of a eukaryotic chromosome: Redesigning and SCRaMbLE-ing yeast.</a> Bioessays. 36 (9), 855-860 (2014).</li><li>Dymond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+genomic+DNA+from+Saccharomyces+cerevisiae.">Preparation of genomic DNA from Saccharomyces cerevisiae.</a> Methods Enzymol. 529, 153-160 (2013).</li><li>Brachmann, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designer+deletion+strains+derived+from+Saccharomyces+cerevisiae+S288C:+a+useful+set+of+strains+and+plasmids+for+PCR-mediated+gene+disruption+and+other+applications.">Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.</a> Yeast. 14 (2), 115-132 (1998).</li></ol>

L.A.M., N.A.P., J.A.M. and J.D.B. have nothing to disclose. R.C. and A.L. are employed by Roche Life Science, U.S. and worked on developing the application with L.A.M. as part of a proof of principle experiment.

Incorporation of real time PCR detection into the PCRTag genotyping assay is an important development for the Sc2.0 project as it enables significantly higher throughput. The previous workflow specified 2.5 &#181;l reactions in 384 well PCR plates, 1.5 hr thermal cycling run time, agarose gel electrophoresis, and manual annotation of the gel.
The workflow presented here, called qPCRTag analysis, overcomes several major bottlenecks. First, a qPCRTag run condenses 4 x 384 well plates into a single experiment that can be processed, start to finish (plate set up plus run time), in about an hour. It is significant to note that the reagent cost per well for qPCRTag analysis is on par with the lower throughput agarose gel-based approach. However, by circumventing gel electrophoresis and manual annotation, the qPCRTag workflow affords substantial savings in time and labor, which are the major advantages. Importantly the qPCRTag workflow is entirely compatible with automation.
A major concern with the use of real time detection as the output of the PCRTag assay is the rate of false positives and false negatives. Since the PCRTag primers were not originally designed for use in real time PCR, it is expected that not all primer pairs will be appropriate for use with this output. To this end it is important to validate the function and specificity of all PCRTag primer pairs to exclude the subset that do not work in the real time-based assay up front. For instance, chromosome 3 PCRTag false positives and negatives are by-and-large reproducible in the real time PCR data (Figures 2 and 3) and these can be excluded from further analyses. Further, primer pairs known to fail (assessed by gel electrophoresis and shown in Figures S6 and S7 of Annaluru et al.1) can also be excluded. This is easily accomplished simply excluding the faulty primer pairs when setting up the acoustic transfer protocol.
Like most high throughput assays, we intend to use real time PCRTag detection as a primary screen to identify transformants that merit further downstream validation. Subsequently, the gold standard for secondary screening will remain PCRTag analysis with gel electrophoresis as the readout. Beyond the application of PCRTag analysis for Sc2.0, combining state-of-the-art nanoscale liquid handling systems with high throughput real time PCR technology enables rapid and automated analysis and has potential to impact many fields. For example, this workflow could be applied to high throughput library screening, infectious disease diagnosis, microbiome analysis, and cutting-edge genome editing approaches attempting to modify multiple loci simultaneously.

Sc2.0, or the Synthetic Yeast Genome Project (<a href="http://www.syntheticyeast.org" target="_blank">www.syntheticyeast.org</a>), has set the goal of designing and building an entirely synthetic eukaryotic genome. Using the highly curated genome sequence of Saccharomyces cerevisiae3 as a starting point, each of the sixteen linear chromosomes has been re-designed to meet a set of design principles that specify maintaining cell fitness, improving genome stability, and increasing genetic flexibility. For instance, destabilizing elements such as repeats are deleted from Sc2.0 chromosomes. All instances of TAG stop codons are re-coded to TAA to &#8216;free up&#8217; a codon in the final strain for the introduction of a non-genetically encoded amino acid. Additionally an inducible evolution system, SCRaMbLE, enabled by the Cre-lox system, permits unprecedented capacity to generate derivative genomes with novel structures4.
Another major design element in the Sc2.0 genome is the introduction of PCRTags, which serve as DNA watermarks to enable tracking of synthetic and wild type DNA. PCRTags are short, re-coded segments in ORFs on synthetic chromosomes; while the PCRTag sequences differ at the DNA level between synthetic and wild type chromosomes, the encoded proteins are identical in amino acid sequence and thus, presumably, function. PCRTag sequences are specifically designed as primer binding sites to facilitate selective amplification (Figure 1A). PCRTag design is carried out using the &#8216;most different&#8217; algorithm in GeneDesign5,6, yielding recoded synthetic sequences that are typically ~60% different than the native sequences (minimum 33%) with melting temperatures between 58 &#176;C and 60 &#176;C and amplicon lengths between 200-500 base pairs2. Recoding is not permitted within the first 100 bp of each ORF, as these regions are known to have special preferences in terms of codon usage7. Together, these design rules favor high performance of almost all PCRTags under a single set of PCR conditions whereby synthetic and wild type PCRTag primer pairs exclusively bind and amplify synthetic and native DNA, respectively (Figure 1B).
<img alt="Figure 1" src="/files/ftp_upload/52941/52941fig1.jpg" /> 
Figure 1: PCRTag schematic. (A) PCRTags are re-coded sequences within open reading frames (ORF) of genes on Sc2.0 chromosomes. (B) Synthetic (SYN) and wild type (WT) PCRTag primers exclusively bind and amplify synthetic and wild type genomic DNA (gDNA), respectively. Shown here is an analysis of a ~30 kb segment of the left arm of chromosome six, testing thirteen PCRTag primer pairs using either WT or semi-synVIL2 gDNA as template. In many cases a single ORF encodes more than one PCRTag. Presence/absence of PCRTag amplicons is assessed via agarose gel electrophoresis. PCRTag amplicons range in size from 200 bp to 500 bp. The faster migrating species at the bottom of the panels are primer dimers.
PCRTag analysis has proven to be an important tool in the assembly of Sc2.0 chromosomes. In a typical experiment 30-50 kb of synthetic DNA, encoding 20-30 PCRTags, is transformed into yeast cells to replace the corresponding wild type DNA1,2,8. PCRTag analysis is then used to identify transformants that encode synthetic but not wild type PCRTags spanning that segment of DNA, or so-called &#8216;winners&#8217;. It is usually necessary to test multiple transformants to identify &#8216;winners&#8217;, so throughput and cost of PCRTag analysis are important considerations. Currently two Sc2.0 chromosomes have been completed (synIII1 and synIXR2), representing less than 10% of the Sc2.0 genome, although more than half of the remaining chromosomes are currently undergoing synthesis and assembly. The scale of PCRTag analysis required for this project is fast outpacing the ability to run gels and manually score the presence of synthetic DNA and absence of wild type DNA.
To improve the throughput of the PCRTag assay we have developed a workflow using real time PCR to circumvent the use of agarose gel electrophoresis. The workflow makes use of a bulk liquid dispenser to distribute qPCR mastermix into each well of a 1,536 multiwell plate, a nanoscale acoustic liquid dispenser to transfer template DNA and primers, and a 1,536 qPCR thermal cycler, allowing us to miniaturize reactions to 500 nl and maximize throughput. Moreover analysis can be automated. This type of high throughput genotyping protocol should be generalizable to any project requiring analysis of many clones at multiple loci.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Yeast gDNA prep</td>
		</tr>
		<tr>
			<td>yeast gDNA</td>
			<td>custom</td>
			<td>custom</td>
			<td>template for real time PCR</td>
		</tr>
		<tr>
			<td>Acid-washed glass beads (0.5mm)</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td>yeast cell lysis</td>
		</tr>
		<tr>
			<td>Ultrapure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
			<td>yeast cell lysis</td>
		</tr>
		<tr>
			<td>5432 Mixer</td>
			<td>Eppendorf</td>
			<td>5432</td>
			<td>yeast cell lysis</td>
		</tr>
		<tr>
			<td>Microcentrifuge 5417R</td>
			<td>Eppendorf</td>
			<td>22621807</td>
			<td>yeast cell lysis</td>
		</tr>
		<tr>
			<td>Qubit 3.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q33216</td>
			<td>gDNA quantification</td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>Life Technologies</td>
			<td>Q32850</td>
			<td>gDNA quantification</td>
		</tr>
		<tr>
			<td>Labcyte 384PP plate</td>
			<td>Labcyte</td>
			<td>P-05525</td>
			<td>gDNA source plate</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>DNA Green Mastermix prep and dispensing</td>
		</tr>
		<tr>
			<td>LightCycler 1536 DNA Green</td>
			<td>Roche</td>
			<td>5573092001</td>
			<td>real time PCR mastermix</td>
		</tr>
		<tr>
			<td>1.5mL Microfuge Tube Holder</td>
			<td>ARI</td>
			<td>EST BD060314-1</td>
			<td>microfuge tube holder for deck of Cobra</td>
		</tr>
		<tr>
			<td>LightCycler 1536 Multiwell Plate</td>
			<td>Roche</td>
			<td>5358639001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cobra liquid handling system</td>
			<td>Art Robbins Instruments</td>
			<td>630-1000-10</td>
			<td>dispense qPCR master mix into 1536 plate</td>
		</tr>
		<tr>
			<td>PCR Plate Spinner</td>
			<td>VWR</td>
			<td>89184-608</td>
			<td>cenrifugation of 1536 plate</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Template and primer dispensing</td>
		</tr>
		<tr>
			<td>PCRTag primers</td>
			<td>IDT</td>
			<td>custom</td>
			<td>premixed forward and reverse, [50uM] each</td>
		</tr>
		<tr>
			<td>TempPlate pierceable sealing foil, sterile</td>
			<td>USA Scientific</td>
			<td>2923-0110</td>
			<td>temporary seal for PCRTag primer plates</td>
		</tr>
		<tr>
			<td>Labcyte LDV 384 well plate</td>
			<td>Labcyte</td>
			<td>LP-0200</td>
			<td>pre-mixed primer source plate</td>
		</tr>
		<tr>
			<td>Echo 550 Liquid Handler</td>
			<td>Labcyte</td>
			<td></td>
			<td>transfer 2.5nL drops of primer and template DNA into 1536 plate</td>
		</tr>
		<tr>
			<td>Plateloc Thermal Microplate Sealer</td>
			<td>Agilent</td>
			<td>G5402-90001</td>
			<td>heat seal for 1536 plate prior to LC1536 run</td>
		</tr>
		<tr>
			<td>Clear Permanent Seal</td>
			<td>Agilent</td>
			<td>24212-001</td>
			<td>optically clear heat seal for LC1536 multiwell plate</td>
		</tr>
		<tr>
			<td>PlateLoc Roche/LightCycler 1536 Plate Stage</td>
			<td>Agilent</td>
			<td>G5402-20008</td>
			<td>can substitute ~2mm thick washers&#160;</td>
		</tr>
		<tr>
			<td>Sorvall Legend XTR</td>
			<td>Thermo Scientfic</td>
			<td>75-004-521</td>
			<td>centrifuge heat sealed 1536 plate</td>
		</tr>
		<tr>
			<td>Industrial Air Compressor</td>
			<td>Jun Air</td>
			<td>1795011</td>
			<td>to run the Echo and Heat Sealer</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Real Time PCR</td>
		</tr>
		<tr>
			<td>LightCycler 1536</td>
			<td>Roche</td>
			<td></td>
			<td>requires 220V outlet</td>
		</tr>
	</tbody>
</table>

qpcrtag analysis - a high throughput, real time pcr assay for sc2.0 genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

We tested synthetic and wild type chromosome 3 PCRTag primer pairs1 with yeast genomic DNA (gDNA) extracted from four different strains. Chromosome 3 has 186 PCRTag primer pairs that span the length of the chromosome (synIII is ~270 kb and wild type chromosome 3 is ~315 kb). To test each of the four strains with both sets of primers, we divided the multiwell plate into four quadrants, one for each type of gDNA, assigning synthetic PCRTag primers to the top half of each quadrant and wild type to the bottom half. Genomic DNA was extracted from the yeast strains, two of which encode wild type chromosome 3 (wild type, synIXR2), while the remaining two encode synthetic chromosome 3 (synIII1, synIII synIXR). qPCR master mix was dispensed into each well of a 1,536 multiwell plate using a bulk liquid dispenser, followed by gDNA and PCRTag primers using the Echo 550. Primers were arrayed identically in each quadrant of the multiwell plate for easy visual comparison. The multiwell plate was then heat sealed with optically clear seal and subjected to real time PCR analysis.
In this qPCRTag experiment we observed, for the most part, amplification as expected, whereby synthetic primers exclusively amplified synthetic DNA and vice versa (Figures 2 and 3). However, we also observed several deviations from the expected pattern, suggesting false negatives and false positives in the dataset. In this experiment, the master control was detected in 100% of wells, indicating the bulk liquid dispenser successfully dispensed mastermix into every well on the plate (data not shown). This rules out one potential source of false negatives. Additionally, some chromosome 3 PCRTags are known to fail (shown in Figures S6 and S7 of Annaluru et al.1), including at least 2 SYN and 1 WT primer pairs; thus these wells can be ignored in each quadrant. True false negatives could arise from a lack of transfer of template gDNA or primers, however in our experience, given the correct calibration of the Echo 550 as well as preparation of gDNA and primers as described, this has not been a major source of error. Overall in this experiment the false negative rate was extremely low for WT primers with WT template (~2%) although somewhat higher for SYN primers with SYN DNA (~8%).
False positives, the detection of signal in wells where SYN primers are mixed with WT gDNA (and vice versa), can arise from cross amplification or primer dimers. Indeed, primer dimers are often visible by gel electrophoresis (Figure 1B) and represent a reasonable source of error. For the application of qPCR, the examination of melt curves can be useful to determine whether different species, such as primer dimers, may be contributing to a signal. Further, performing a control experiment whereby primers are dispensed in the absence of template DNA may help identify primers with a propensity to dimerize. Cross amplification can be observed by gel electrophoresis, in particular if too many PCR cycles are performed or if the annealing temperature is too low. We have tried to minimize the number of false positives due to cross amplification by optimizing both of these parameters for the qPCRTag protocol. Finally, examining the crossing point (Cp) values for each well can help identify primers that are not suited to real time-based detection (Figure 3, e.g., Bb22, Fb22, Bb46, Fb46). Overall in this experiment the false positive rate was low for SYN primers with WT template (~5%) and higher for WT primers with SYN template (~10%).
<img alt="Figure 2" src="/files/ftp_upload/52941/52941fig2.jpg" /> 
Figure 2: Plate heat map displaying presence/absence call for a qPCRTag experiment. Four different types of genomic DNA (quadrants separated by solid white lines) were subjected to PCRTag analysis using synthetic (SYN) and wild type (WT) chromosome 3 PCRTag primers (dashed white lines to separate SYN (top) and WT (bottom) in each quadrant). WT and synIXR gDNA encode wild type chromosome 3, yielding amplification with WT PCRTag primers. synIII and synIII synIXR gDNA encode synthetic chromosome 3, yielding amplification with SYN PCRTag primers. Primers are arrayed according to their left-to-right chromosomal positioning and positioned identically in the four quadrants for comparison. <a href="https://www.jove.com/files/ftp_upload/52941/52941fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52941/52941fig3.jpg" /> 
Figure 3: Plate heat map displaying crossing point (Cp) value for a qPCRTag experiment. This is the same dataset as in Figure 2 and the plate layout is therefore identical. N/A refers to &#8216;no amplification&#8217;. <a href="https://www.jove.com/files/ftp_upload/52941/52941fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping at JoVE.com

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

qpcrtag-analysis-high-throughput-real-time-pcr-assay-for-sc20

Research

JoVE Journal

Biology

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

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.

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 ...