This research was funded by a grant from the Bill &#38; Melinda Gates Foundation through the Grand Challenges in Global Health Initiative.

<ol>
	<li>Yoon, J. -. Y., Kim, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-a-Chip+Pathogen+Sensors+for+Food+Safety.">Lab-on-a-Chip Pathogen Sensors for Food Safety.</a> Sensors. 12 (8), 10713-10741 (2012).</li><li>Quintero-Betancourt, W., Peele, E. R., Rose, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptosporidium+parvum+and+Cyclospora+cayetanensis:+A+review+of+laboratory+methods+for+detection+of+these+waterborne+parasites.">Cryptosporidium parvum and Cyclospora cayetanensis: A review of laboratory methods for detection of these waterborne parasites.</a> Journal of Microbiological Methods. 49 (3), 209-224 (2002).</li><li>Sedlak, R. H., Jerome, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+diagnostics+in+the+era+of+digital+polymerase+chain+reaction.">Viral diagnostics in the era of digital polymerase chain reaction.</a> Diagnostic Microbiology and Infectious Disease. 75 (1), 1-4 (2013).</li><li>Asiello, P. J., Baeumner, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+isothermal+nucleic+acid+amplification,+a+review.">Miniaturized isothermal nucleic acid amplification, a review.</a> Lab on a Chip. 11 (8), 1420-1430 (2011).</li><li>Piepenburg, O., Williams, C. H., Stemple, D. L., Armes, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+detection+using+recombination+proteins.">DNA detection using recombination proteins.</a> PLoS Biology. 4 (7), 1115-1121 (2006).</li><li>Kr&#245;lov, K., Frolova, 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=Sensitive+and+Rapid+Detection+of+Chlamydia+trachomatis+by+Recombinase+Polymerase+Amplification+Directly+from+Urine+Samples.">Sensitive and Rapid Detection of Chlamydia trachomatis by Recombinase Polymerase Amplification Directly from Urine Samples.</a> The Journal of Molecular Diagnostics JMD. 16 (1), 127-135 (2014).</li><li>Santiago-Felipe, S., Tortajada-Genaro, L. a., Puchades, R., Maquieira, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinase+polymerase+and+enzyme-linked+immunosorbent+assay+as+a+DNA+amplification-detection+strategy+for+food+analysis.">Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.</a> Analytica Chimica Acta. 811 (1), 81-87 (2014).</li><li>Kersting, S., Rausch, V., Bier, F. F., von Nickisch-Rosenegk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+Plasmodium+falciparum+with+isothermal+recombinase+polymerase+amplification+and+lateral+flow+analysis.">Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis.</a> Malaria Journal. 13 (1), 99 (2014).</li><li>Crannell, Z. 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=Nucleic+Acid+test+to+diagnose+cryptosporidiosis:+lab+assessment+in+animal+and+patient+specimens.">Nucleic Acid test to diagnose cryptosporidiosis: lab assessment in animal and patient specimens.</a> Analytical Chemistry. 86 (5), 2565-2571 (2014).</li><li>Rohrman, B. A., Richards-Kortum, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+paper+and+plastic+device+for+performing+recombinase+polymerase+amplification+of+HIV+DNA.">A paper and plastic device for performing recombinase polymerase amplification of HIV DNA.</a> Lab on a Chip. 12 (17), 3082 (2012).</li><li>Loo, J. F. C., Lau, P. M., Ho, H. P., Kong, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+aptamer-based+bio-barcode+assay+with+isothermal+recombinase+polymerase+amplification+for+cytochrome-c+detection+and+anti-cancer+drug+screening.">An aptamer-based bio-barcode assay with isothermal recombinase polymerase amplification for cytochrome-c detection and anti-cancer drug screening.</a> Talanta. 115 (1), 159-165 (2013).</li><li>Euler, M., 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=Development+of+a+panel+of+recombinase+polymerase+amplification+assays+for+detection+of+biothreat+agents.">Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.</a> Journal of Clinical Microbiology. 51 (4), 1110-1117 (2013).</li><li>Abd El Wahed, 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=A+portable+reverse+transcription+recombinase+polymerase+amplification+assay+for+rapid+detection+of+foot-and-mouth+disease+virus.">A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus.</a> PloS One. 8 (8), e71642 (2013).</li><li>Escadafal, C., 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=Rapid+Molecular+Assays+for+the+Detection+of+Yellow+Fever+Virus+in+Low-Resource+Settings.">Rapid Molecular Assays for the Detection of Yellow Fever Virus in Low-Resource Settings.</a> PLoS Neglected Tropical Diseases. 8 (3), e2730 (2014).</li><li>Hutson, S. L., 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=T.+gondii+RP+Promoters+&amp;+Knockdown+Reveal+Molecular+Pathways+Associated+with+Proliferation+and+Cell-Cycle+Arrest.">T. gondii RP Promoters &amp; Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest.</a> PLoS ONE. 5 (11), 15 (2010).</li><li>Segall, H. I., Yoo, E., Sutton, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+detection+of+artificial+replication-competent+lentivirus+of+altered+host+range.">Characterization and detection of artificial replication-competent lentivirus of altered host range.</a> Molecular Therapy. 8 (1), 118-129 (2003).</li><li>Crannell, Z. A., Rohrman, B. A., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+HIV-1+DNA+using+Real-Time+Recombinase+Polymerase+Amplification.">Quantification of HIV-1 DNA using Real-Time Recombinase Polymerase Amplification.</a> Analytical Chemistry. 86 (12), (2014).</li><li>Sollis, K. 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=Systematic+review+of+the+performance+of+HIV+viral+load+technologies+on+plasma+samples.">Systematic review of the performance of HIV viral load technologies on plasma samples.</a> PloS one. 9 (2), e85869 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. , 1-166 (2011).</li></ol>

The authors declare they have no competing financial interests.

In order to obtain meaningful quantification data using the MATLAB algorithm, the user must select appropriate input values when prompted. After initiating each script in Sections 5 and 6, all input variables are automatically solicited in the command window and outputs are automatically generated. In Section 5.7 the user is prompted to select a slope threshold. The value of the slope threshold affects the square of the correlation coefficient (r2) of the fit. When using raw fluorescence data exported from a thermal cycler, values between 2.0 and 5.0 tend to yield a high r2 coefficient. In Section 5.8 the user must designate the number of standard deviations above the background to set the positive threshold. To score a sample as positive or negative, the script automatically determines the difference &#916;sample between the maximum and minimum fluorescence for each sample using the raw fluorescence data exported from the thermal cycler. It calculates the average difference &#916;background and standard deviation &#963;background for all no-target control samples. A sample is considered positive if &#916;sample is more than z &#215; &#963;background above &#916;background. In Section 5.10 the user decides whether to use the default threshold or set a new threshold. If the user wishes to set a new threshold, determine the new threshold experimentally by performing 3 experiments each containing 12 RPA reactions without any HIV-1 DNA present. Set the threshold at the average increase in fluorescence intensity from these experiments plus 3 standard deviations. After completing Section 6, the script JoVE_qRPA_validation_and_quantification.m automatically returns the estimated DNA concentration for each qRPA reaction (in log10 copies). If the script determines that no HIV-1 DNA was present in the sample, the estimated concentration is listed as either &#8220;Negative for HIV&#8221; or &#8220;Invalid,&#8221; depending on whether the fluorescent signal for the internal positive control exceeded the threshold (z &#215; &#963;background + &#916;background). If the user is validating the standard curve with known sample concentrations, the script will also return an additional table similar to that of Table 1.
In order to develop a real-time RPA assay that provides accurate quantification, experimental consistency is crucial. For example, use the same primer and probe aliquots for both the standard curve and validation experiments. Also avoid freeze-thaw cycles by storing the primer and probe aliquots at 4 &#176;C between experiments rather than -20 &#176;C. The template aliquots used for the standard curve and validation experiments are stored in the same manner. RPA enzyme pellets and reagents from the same lot are used according to the manufacturer recommendations. Lastly, because RPA lacks true &#8216;cycles&#8217; to precisely control the rate of amplification, standardization of user steps is absolutely imperative. When assembling reactions, the user must always follow the same steps in the same order, spending approximately the same amount of time on each step. Reactions must always be mixed gently with a new pipette tip, and bubbles must always be eliminated. Before amplification, reactions must be held at a consistent temperature, and the thermal cycler or microscope software must always be prepared before loading reactions to avoid any amplification at sub-optimal temperatures that could influence quantification. Any variation in the initial reaction conditions may lead to inconsistency in experimental outcomes.
When using a microscope to collect data, additional variables must be controlled to minimize variation in fluorescence intensity. All reactions must be placed in the same region on the stage warmer, and the microscope must be focused on the same region of the well for every sample. Even if these practices are followed, fluorescence data collected on a microscope may exhibit variability due to local bright spots that naturally form during RPA reactions, bubble formation within the reaction chambers, or photobleaching resulting from repeated exposure to excitation light. The influence of these variables is evident in fluorescence data collected on the microscope (Figures 4A and 4B), which demonstrate baseline variability, peaks, and troughs. These features are absent from the fluorescence data collected on the thermal cycler (Figures 3A and 3B). Ultimately, data collection on the microscope is for proof-of-principle purposes only and the final assay will be implemented on a field-operable fluorescence reader with more precise geometry and software control that minimizes these variables.
Another important aspect of the qRPA assay development process is consistency in data processing. The protocol described in the methods section uses scripts to process raw fluorescence data (stored in a spreadsheet file) collected from a thermal cycler or microscope. All experiments used to build the standard curve must be formatted identically. When using a thermal cycler to collect data, the same plate layout must be used, and data from wells that do not contain RPA reactions must not be exported. When using a microscope to collect data, the format of the data must match the format of the data automatically exported from the thermal cycler. For example, the no-target-control data must be in cells C2:C61, and data for increasing template concentrations must be in cells D2:D61, E2:E61, etc. If there are multiple replicates of each concentration in an experiment, the 2nd replicate dilution series must be ordered left to right from no target control (NTC) to highest concentration and saved in the columns immediately to the right of the 1st replicate dilution series. For example, in the plate layout used in Section 1.2 with 2 replicates for each sample, fluorescence data for the 1st replicate of each sample in the dilution series must be saved in cells C2:H61 and fluorescence data for the 2nd replicate of each sample in the dilution series must be saved in cells I2:N61. For the plate layout used in Section 1.2, this is the default formatting when exporting data from the thermal cycler software to a spreadsheet.
Representative data provided from HIV-1 qRPA experiments demonstrate proof-of-concept support that RPA may be used for quantification of nucleic acid concentration in unknown samples. Clinically useful HIV-1 viral load tests have a clinical range of at least 4 orders of magnitude, a precision of 0.5 log10 copies, and a limit-of-detection of at least 200 copies19,20. The HIV-1 DNA assay described meets these criteria and is most accurate at low concentrations, as shown in Table 1. Therefore, with the inclusion of a reverse transcriptase step, these results suggest that an HIV-1 RT-RPA assay may have the potential to measure HIV-1 viral load in clinical samples. When developing a qRPA assay, adjusting the algorithm parameters may tune the sensitivity and linear dynamic range depending on clinical needs. Figure 6 shows that adjusting z (a parameter that determines the threshold for positive samples) can influence the sensitivity and accuracy at low and high target concentrations. Furthermore, it may be possible to increase the resolution and accuracy of quantification by incubating reactions at a lower temperature or using less magnesium acetate, thereby decreasing the rate of amplification.
This proof of concept qRPA assay can be used to quantify the concentration of samples containing HIV-1 DNA. The qRPA assay described in this manuscript includes detailed instructions on how to assemble real-time RPA reactions, develop and screen an IPC, and process raw fluorescence data to build a standard curve that can be used to quantify unknown samples. With the detailed instructions included, this protocol may be adapted to quantify DNA concentration in a wide variety of samples.

Quantitative nucleic acid amplification is an important technique for detection of environmental, foodborne, and water-borne pathogens as well as for clinical diagnostics. Real-time quantitative polymerase chain reaction (qPCR) is the gold standard method for sensitive, specific, and quantitative detection of nucleic acids, e.g., for HIV-1 viral load testing, detection of bacterial pathogens, and screening for many other organisms1&#8211;3. During real-time qPCR, primers amplify pathogen DNA in cycles, and a fluorescent signal is generated that is proportional to the amount of amplified DNA in the sample at each cycle. A sample containing an unknown concentration of pathogen DNA may be quantified using a standard curve that relates the initial DNA concentration of standard samples and the time at which the fluorescent signal reaches a certain threshold (i.e., the cycle threshold, or CT).
Because real-time qPCR requires expensive thermal cycling equipment and several hours to receive results, alternative isothermal amplification techniques, such as recombinase polymerase amplification (RPA), have been developed4. These platforms generally provide results faster and amplify nucleic acids at a lower, single temperature, which may be accomplished with less expensive, simpler equipment. RPA, which is particularly attractive for point-of-care applications, amplifies DNA in minutes, requires a low amplification temperature (37 &#176;C), and remains active in the presence of impurities5,6. RPA assays have been developed for a wide range of applications, including food analysis, pathogen detection, cancer drug screening, and detection of biothreat agents7&#8211;12. However, use of RPA for quantification of nucleic acids has been limited13,14.
In previous work, it was shown that real-time quantitative RPA (qRPA) is feasible15. Here, a more detailed protocol is provided for using real-time quantitative RPA to quantify unknown samples using a standard curve, a method that is analogous to quantification using qPCR. This protocol describes how to perform an RPA reaction on a thermal cycler to detect HIV-1 DNA as a proof-of-concept, as well as how to develop an internal positive control (IPC) to ensure the system is functioning properly. Data collection using a thermal cycler or microscope and data analysis for constructing a standard curve using training data is also detailed. Finally, the method for quantifying unknown samples using the standard curve with a custom script is demonstrated. This qRPA technique enables quantification of samples with unknown concentrations and has many advantages over traditional real-time qPCR.

<table border="0" cellpadding="0" cellspacing="0" width="1426">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:443px;">qRPA Assay</td>
			<td style="width:199px;"></td>
			<td style="width:195px;"></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Supply</td>
			<td>Vendor</td>
			<td>Part number</td>
			<td style="width:589px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HIV-1 forward primer</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td style="width:589px;">5&#8217;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G-3&#8217;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HIV-1 reverse primer</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td style="width:589px;">5&#8217;-CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G-3&#8217;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HIV-1 probe</td>
			<td>BioSearch Technologies&#160;</td>
			<td>custom DNA oligos</td>
			<td style="width:589px;">5&#8217;- TGC TAT TAT GTC TAC TAT TCT TTC CCC [SIMA/HEX] GC [THF] C [dT-BHQ1] GTA CCC CCC AAT CCC C -3&#8217;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IPC probe</td>
			<td>BioSearch Technologies&#160;</td>
			<td>custom DNA oligos</td>
			<td style="width:589px;">5&#8217;-AGG TAG TGA CAA GAA ATA ACA ATA CAG GAC [FAM] T [THF] T [dT-BHQ1] GGT TTT GTA ATT GGA A -3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPA exo reagents (pellets, rehydration buffer, magnesium acetate</td>
			<td>TwistDx</td>
			<td>TwistAmp exo</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR tube strips</td>
			<td>BioRad</td>
			<td>TLS0801</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR flat cap tube strips</td>
			<td>BioRad</td>
			<td>TCS0803</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-seal adhesive</td>
			<td>BioRad</td>
			<td>558/MJ&#160;</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">HIV-1 target (pHIV-IRES- eYFP&#916;Env&#916;Vif&#916;Vpr)</td>
			<td></td>
			<td></td>
			<td style="width:589px;">custom plasmid, see: Segall, H. I., Yoo, E. &#38; Sutton, R. E. Characterization and detection of artificial replication-competent lentivirus of altered host range. Molecular Therapy 8, 118&#8211;129, doi:10.1016/S1525-0016(03)00134-5 (2003).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human male genomic DNA</td>
			<td>Applied Biosystems</td>
			<td>360486</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well cold-block</td>
			<td>Cole Parmer</td>
			<td>EW-36700-48</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermal cycler</td>
			<td>BioRad</td>
			<td>CFX96</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MiniFuge</td>
			<td>VWR</td>
			<td>93000-196</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex</td>
			<td>VWR</td>
			<td>58816-121</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris buffer 1.0 M, pH 8.0</td>
			<td>EMD Millipore</td>
			<td>648314</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA 0.5 M, pH 8.0</td>
			<td>Promega</td>
			<td>V4321</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease free water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPC Development</td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Supply</td>
			<td>Vendor</td>
			<td>Part number</td>
			<td style="width:589px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Cryptosporidium parvum IPC template</td>
			<td>Waterborne Inc</td>
			<td>P102C</td>
			<td style="width:589px;">It is also possible to order a double stranded synthetic target from IDT if the user is unequipped to work with C. parvum (a BSL-2 infectious agent). PCR and RPA primers for C. parvum were designed using GenBank accession number AF115377.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR long forward primer</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td>5&#8217;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G/ ATC TAA GGA AGG CAG CAG GC-3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR long reverse primer</td>
			<td>Integrated DNA Technologies</td>
			<td>custom DNA oligos</td>
			<td>5&#8217;- CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G/ TGC TGG AGT ATT CAA GGC ATA -3&#8217;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phusion High-Fidelty DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qiaquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TAE 10X buffer</td>
			<td>EMD Millipore</td>
			<td>574797</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9539</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Experiments</td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Supply</td>
			<td>Vendor</td>
			<td>Part number</td>
			<td style="width:589px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Upright fluorescence microscope</td>
			<td>Zeiss</td>
			<td>Zeiss Imager.J1</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stage heater</td>
			<td>Bioscience Tools</td>
			<td>TC-GSH</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1-Channel Precision High Stability Temperature Controller</td>
			<td>Bioscience Tools</td>
			<td>TC-1100S</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAM/GFP filter cube</td>
			<td>Zeiss</td>
			<td>filter set 38 (000000-1031-346)</td>
			<td style="width:589px;">excitation BP 470/40 nm, emission BP 520/50 nm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEX filter cube</td>
			<td>Chroma</td>
			<td>49014</td>
			<td style="width:589px;">excitation BP 530/30 nm, emission BP 575/40 nm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laser cutter</td>
			<td>Engraver&#39;s Network</td>
			<td>VLS3.60</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1/8&#34; black acrylic</td>
			<td>McMaster Carr</td>
			<td>8505K113</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mm clear acrylic</td>
			<td>McMaster Carr</td>
			<td>PD-72268940&#160;</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Super glue</td>
			<td>Office Depot</td>
			<td>Duro super glue&#160;</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR grade mineral oil</td>
			<td>Sigma Aldrich</td>
			<td>M8662-5VL</td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Data Analysis</td>
			<td></td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software</td>
			<td>Vendor</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB</td>
			<td>MATLAB</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB script: &#34;JoVE_qRPA_standard_curve.m&#8221;</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB script: &#34;JoVE_qRPA_validation_and_quantification.m&#8221;</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MATLAB script: &#34;JoVE_real_time_intensity_to_excel.m&#8221;</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adobe Illustrator</td>
			<td>Adobe</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JoVE_qRPA_well.ai</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JoVE_qRPA_base.ai</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AxioVision software</td>
			<td>Zeiss</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">JoVE_AxioVision_Script.ziscript</td>
			<td>Included in SI</td>
			<td></td>
			<td style="width:589px;"></td>
		</tr>
	</tbody>
</table>

development of a quantitative recombinase polymerase amplification assay with an internal positive control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Before selecting a sequence to serve as the IPC in qRPA experiments with target (HIV-1) DNA, internal positive control (IPC) candidates are generated and screened for their ability to amplify in qRPA reactions without HIV-1 DNA present. IPC candidates are longer than the target (HIV-1) DNA to prevent IPC formation from out-competing HIV-1 amplicon formation. As shown in Figure 2A, the generation of two C. parvum IPC candidates was verified by the presence of 415 and 435 bp bands using gel electrophoresis. In qRPA reactions, the shorter IPC candidate exhibited little amplification (Figure 2B), while the longer candidate consistently amplified when a total of 104 and 105 copies were present (Figure 2C). Thus, the longer candidate was chosen to be the IPC for HIV-1 qRPA experiments.
Real-time qRPA may be performed using the target of interest alone or using both the target and the IPC. Figure 3 shows an experiment on the thermal cycler using both HIV-1 DNA and the C. parvum IPC. In this experiment, 2.6 x 104 copies of IPC DNA were added to each reaction, a concentration close to the IPC limit of detection, to avoid affecting the limit of detection of HIV-1 target DNA. As demonstrated in Figure 3A, which displays HEX fluorescence data corresponding to real-time HIV-1 DNA generation, the time at which detectable amplification begins is inversely proportional to the initial target concentration. Thus, amplification is apparent earlier for high concentrations of HIV-1 DNA and later for low concentrations of HIV-1 DNA. In contrast, detectable amplification of IPC DNA begins at approximately the same time because the starting IPC concentration is the same in all samples, as shown in Figure 3B, which displays FAM fluorescence data corresponding to IPC DNA generation during the same experiment. The rate of fluorescence generation from the IPC is inversely proportional to the concentration of HIV-1 DNA due to competition during amplification.
With the goal of developing a field-operable fluorescence reader with qRPA reactions, qRPA experiments may be performed on an upright fluorescence microscope with a stage heater and 1-Channel Precision High Stability temperature controller. Figure 4A and 4B display HEX and FAM fluorescence data collected on a microscope. Data collected on the microscope using laser-cut chips demonstrate slight variability in baseline fluorescence and crests and troughs that may be due to photobleaching. However, the script determines the cycle threshold using the rate of change of fluorescence during the initial amplification period, which is unaffected by baseline fluorescence or variability in fluorescence after the initial amplification has occurred. As seen in Figure 4C, the standard curve built from this experiment has an r2 coefficient of 0.990. Notably, the IPC is amplified only for low concentrations of HIV-1 DNA. Although this behavior differs from experiments performed on the thermal cycler, all samples are still classified as valid using this method.
Data from multiple experiments using known target concentrations may be compiled to construct a standard curve, which may then be used to assess assay performance or quantify samples with unknown concentrations. Figure 5 shows the data from five experiments and an exponential standard curve generated using the script &#8220;JoVE_qRPA_standard_curve.m&#8221;, relating the initial HIV-1 target concentration to the time at which detectable amplification began. Figure 5 used 5 separate experiments, each containing 2 qRPA reactions at each template concentration to build a standard curve. Note that the exponential fit has a high r2 coefficient of 0.959. The standard curve was then used to predict the concentrations of additional samples with known concentrations for assay validation using the script &#8220;JoVE_qRPA_validation_and_quantification.m&#8221; script. Table 1 shows the quantification results for 5 additional experiments using the standard curve in Figure 5. Note that the algorithm correctly classified all samples containing HIV-1 DNA as positive and 9 out of 10 of the no-target-control samples as negative. In addition, the average predicted concentration was within one order of magnitude of the correct concentration and the standard deviation for predicted concentrations was less than 0.5 log10 (copies per reaction).
<img alt="Figure 1" src="/files/ftp_upload/52620/52620fig1highres.jpg" /> 
Figure 1: Block diagram of quantitative RPA DNA targets. The qRPA assay amplifies two DNA sequences flanked by identical sequences to which primers bind (&#8220;RPA forward&#8221; and &#8220;RPA reverse&#8221;). The amplified sequences are 1) a sequence within the HIV-1 genome (&#8220;HIV-1&#8221;) that is detected with the &#8220;HIV-1 probe&#8221; and 2) an internal positive control sequence (&#8220;IPC (C. parvum)&#8221;) included in each reaction that is detected with the &#8220;IPC probe.&#8221; The IPC was generated via PCR using the Cryptosporidium parvum genome as a template and primers (&#8220;PCR forward&#8221; and &#8220;PCR reverse&#8221;) that contain a region complementary to C. parvum (purple) and a region complementary to HIV-1 (blue). Figure originally published in Analytical Chemistry 2014, reprinted here with permission9.
<img alt="Figure 2" src="/files/ftp_upload/52620/52620fig2.jpg" /> 
Figure 2: Screening of IPC candidates. (A) Two IPC candidates (&#8216;Forward Short&#8217; and &#8216;Forward Long&#8217;) and a known PCR positive control sequence were generated using PCR and visualized on an agarose gel, where 415 bp and 435 bp bands were visible. (B) The short IPC candidate exhibited little amplification during real-time RPA, while (C) the longer IPC candidate amplified consistently when a total of 104 and 105 copies were present. Figure originally published in Analytical Chemistry 2014, reprinted here with permission9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52620/52620fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52620/52620fig3highres.jpg" /> 
Figure 3: Typical raw fluorescence data generated during co-amplification of HIV-1 DNA and IPC on a thermal cycler. (A) For HIV-1, the onset of detectable amplification, shown by an apparent increase in HEX fluorescence, occurs earlier for high concentrations of HIV-1 DNA. (B) For the IPC, the onset of detectable amplification, shown by an apparent increase in FAM fluorescence, occurs at approximately the same time regardless of the HIV-1 DNA concentration. However, the rate of FAM fluorescence generation is inversely proportional to the amount of HIV-1 DNA present due to competition. Figure originally published in Analytical Chemistry 2014, reprinted here with permission9.
<img alt="Figure 4" src="/files/ftp_upload/52620/52620fig4highres.jpg" /> 
Figure 4: Typical raw fluorescence data generated during co-amplification of HIV-1 DNA and IPC on a microscope. (A) Similar to data generated on the thermal cycler, the onset of detectable HIV-1 amplification, shown by an apparent increase in HEX fluorescence, occurs earlier for high concentrations of HIV-1 DNA. (B) IPC amplification on the microscope, shown by FAM fluorescence, is apparent for low HIV-1 DNA concentrations but not always apparent in samples with high HIV-1 DNA concentrations. (C) A standard curve can be built using the JoVE_standard_curve.m scripts that yields a high r2 coefficient.
<img alt="Figure 5" src="/files/ftp_upload/52620/52620fig5highres.jpg" /> 
Figure 5: Typical standard curve for HIV-1. Using the JoVE_standard_curve.m script, raw HEX fluorescence data generated during HIV-1 amplification is processed to build a standard curve, which may be used to predict the HIV-1 DNA concentration of unknown samples. This standard curve (z = 1) was generated using data from 5 experiments, in which 6 concentrations were tested using 2 replicates at each concentration. All concentrations are given in log10 copies. Figure originally published in Analytical Chemistry 2014, reprinted here with permission9.
<img alt="Figure 6" src="/files/ftp_upload/52620/52620fig6highres.jpg" /> 
Figure 6: Algorithm tunability. Adjusting the algorithm parameters changes the standard curve used to predict the concentration of unknown samples. The JoVE_validation_and_quantification.m script was used to predict the concentration of unknown samples using z = 1 (red) and z = 5 (blue). All concentrations are given in log10 copies. Predictions are more accurate for low DNA concentrations when z = 1; predictions are more accurate for high DNA concentrations when z = 5. By adjusting the algorithm parameters, the qRPA standard curve may be tuned according to clinical needs. Figure originally published in Analytical Chemistry 2014, reprinted here with permission9.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sample Concentration</td>
			<td>Average Predicted Concentration</td>
			<td>Standard Deviation</td>
			<td>Percent of Samples Identified as Positive</td>
		</tr>
		<tr>
			<td>No target controls</td>
			<td>0.1</td>
			<td>0.3</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.9</td>
			<td>0.1</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>2</td>
			<td>2.2</td>
			<td>0.5</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>3</td>
			<td>3</td>
			<td>0.1</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>4</td>
			<td>3.7</td>
			<td>0.1</td>
			<td>100%</td>
		</tr>
		<tr>
			<td>5</td>
			<td>4.2</td>
			<td>0.2</td>
			<td>100%</td>
		</tr>
	</tbody>
</table>
Table 1: HIV-1 qRPA assay validation. Using the standard curve in Figure 4, the JoVE_validation_and_quantification.m script was used to predict the concentrations (in log10 copies) of samples for 5 additional experiments. Table originally published in Analytical Chemistry 2014, reprinted here with permission9.

Watch this Scientific Journal Video about Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control at JoVE.com

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

development-quantitative-recombinase-polymerase-amplification-assay

Nanyang Technological University

Research

JoVE Journal

Biology

13.5K Views.  Rice University. Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

Video: Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Molecular Evolution of the Tre Recombinase

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of "molecular surgery" and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

Interview: HIV-1 Proviral DNA Excision Using an Evolved Recombinase

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies primarily target virus enzymes or virus-cell fusion, suppressing the viral life cycle without eradicating the infection. Since the integrated provirus is not targeted by these approaches, new resistant strains of HIV-1 may emerge. Here, we report that the engineered recombinase Tre (see Molecular evolution of the Tre recombinase , Buchholz, F., Max Planck Institute for Cell Biology and Genetics, Dresden) efficiently excises integrated HIV-1 proviral DNA from the genome of infected cells. We produced loxLTR containing viral pseudotypes and infected HeLa cells to examine whether Tre recombinase can excise the provirus from the genome of HIV-1 infected human cells. A virus particle-releasing cell line was cloned and transfected with a plasmid expressing Tre or with a parental control vector. Recombinase activity and virus production were monitored. All assays demonstrated the efficient deletion of the provirus from infected cells without visible cytotoxic effects. These results serve as proof of principle that it is possible to evolve a recombinase to specifically target an HIV-1 LTR and that this recombinase is capable of excising the HIV-1 provirus from the genome of HIV-1-infected human cells.
Before an engineered recombinase could enter the therapeutic arena, however, significant obstacles need to be overcome. Among the most critical issues, that we face, are an efficient and safe delivery to targeted cells and the absence of side effects.

Current HIV-1 strategies act to suppress the viral life cycle but do not effectively eradicate infection. Here, we demonstrate that an engineered recombinase can efficiently excise integrated HIV-1 proviral DNA from the genome of infected cells.

HIV-1 integrates into the host chromosome of infected cells and persists as a provirus flanked by long terminal repeats. Current treatment strategies ...

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 ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

Analyzing and Building Nucleic Acid Structures with 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures. Protocol 1 lists the set of instructions needed to download and install the software. This is followed, in Protocol 2, by the analysis of a nucleic acid structure, including the assignment of base pairs and the determination of rigid-body parameters that describe the structure and, in Protocol 3, by a description of the reconstruction of an atomic model of a structure from its rigid-body parameters. The most recent version of 3DNA, version 2.1, has new features for the analysis and manipulation of ensembles of structures, such as those deduced from nuclear magnetic resonance (NMR) measurements and molecular dynamic (MD) simulations; these features are presented in Protocols 4 and 5. In addition to the 3DNA stand-alone software package, the w3DNA web server, located at <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a>, provides a user-friendly interface to selected features of the software. Protocol 6 demonstrates a novel feature of the site for building models of long DNA molecules decorated with bound proteins at user-specified locations.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic acid structures. This article presents detailed protocols for a subset of new and popular features available in 3DNA, applicable to both individual structures and ensembles of related structures.

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...