Name: two-step reverse transcription droplet digital pcr protocols for sars-cov-2 detection and quantification
Uploaded: 2021-03-31T13:30:00
Description: Watch this Scientific Journal Video about Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification at JoVE.com

This research was funded by Megaproject of Infectious Disease Control from Ministry of Health of China, grant number 2017ZX10302301-005 and Sino-Africa Joint Research Center, grant number SAJC201605.

Ethical statement 
Wuhan Institute of Virology (WHIOV) is among the labs and institutes approved by China CDC of Wuhan city to conduct research on SARS-CoV-2 and detect COVID-19 from clinical samples. Research on developing new diagnostic techniques for COVID-19 using clinical samples has also been approved by the ethical committee of Wuhan Institute of Virology (2020FCA001).
1. Sample processing workflow (Figure 1A)
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<ol>
	<li>Nyaruaba, R., Mwaliko, C., Kering, K. K., Wei, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+digital+PCR+applications+in+the+tuberculosis+world.">Droplet digital PCR applications in the tuberculosis world.</a> Tuberculosis. 117, 85-92 (2019).</li><li>Baker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR+hits+its+stride.">Digital PCR hits its stride.</a> Nature Methods. 9 (6), 541-544 (2012).</li><li>Quan, P. L., Sauzade, M., Brouzes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dPCR:+a+technology+review.">dPCR: a technology review.</a> Sensors. 18 (4), 1271 (2018).</li><li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR.">Digital PCR.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (16), 9236 (1999).</li><li>Kuypers, J., 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=Applications+of+digital+PCR+for+clinical+microbiology.">Applications of digital PCR for clinical microbiology.</a> Journal of Clinical Microbiology. 55 (6), 1621 (2017).</li><li>Li, H., 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=Application+of+droplet+digital+PCR+to+detect+the+pathogens+of+infectious+diseases.">Application of droplet digital PCR to detect the pathogens of infectious diseases.</a> Bioscience Reports. 38 (6), (2018).</li><li>Liu, X., 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=Analytical+comparisons+of+SARS-COV-2+detection+by+qRT-PCR+and+ddPCR+with+multiple+primer/probe+sets.">Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets.</a> Emerging Microbes &amp; Infections. 9 (1), 1175-1179 (2020).</li><li>Suo, T., 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=ddPCR:+a+more+accurate+tool+for+SARS-CoV-2+detection+in+low+viral+load+specimens.">ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load specimens.</a> Emerging Microbes &amp; Infections. 9 (1), 1259-1268 (2020).</li><li>Liu, Y., 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=Aerodynamic+analysis+of+SARS-CoV-2+in+two+Wuhan+hospitals.">Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals.</a> Nature. 582 (7813), 557-560 (2020).</li><li>Lv, 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=Detection+of+SARS-CoV-2+RNA+residue+on+object+surfaces+in+nucleic+acid+testing+laboratory+using+droplet+digital+PCR.">Detection of SARS-CoV-2 RNA residue on object surfaces in nucleic acid testing laboratory using droplet digital PCR.</a> Science of The Total Environment. 742, 140370 (2020).</li><li>Yu, F., 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=Quantitative+detection+and+viral+load+analysis+of+SARS-CoV-2+in+infected+patients.">Quantitative detection and viral load analysis of SARS-CoV-2 in infected patients.</a> Clinical Infectious Diseases. 71 (15), 793-798 (2020).</li><li>Scutari, R., 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=Long-term+SARS-CoV-2+infection+associated+with+viral+dissemination+in+different+body+fluids+including+bile+in+two+patients+with+acute+cholecystitis.">Long-term SARS-CoV-2 infection associated with viral dissemination in different body fluids including bile in two patients with acute cholecystitis.</a> Life. 10 (11), 302 (2020).</li><li>Nyaruaba, R., 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+field-deployable+RT-qPCR+workflow+for+COVID-19+detection.">Development of a field-deployable RT-qPCR workflow for COVID-19 detection.</a> Preprints. , (2020).</li><li>Whale, A. S., Huggett, J. F., Tzonev, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+multiplexing+with+digital+PCR.">Fundamentals of multiplexing with digital PCR.</a> Biomolecular Detection and Quantification. 10, 15-23 (2016).</li><li>Dobnik, D., &#352;tebih, D., Blejec, A., Morisset, D., &#381;el, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+quantification+of+four+DNA+targets+in+one+reaction+with+Bio-Rad+droplet+digital+PCR+system+for+GMO+detection.">Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.</a> Scientific Reports. 6 (1), 35451 (2016).</li><li>Nyaruaba, R., 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+and+evaluation+of+a+single+dye+duplex+droplet+digital+PCR+assay+for+the+rapid+detection+and+quantification+of+mycobacterium+tuberculosis.">Development and evaluation of a single dye duplex droplet digital PCR assay for the rapid detection and quantification of mycobacterium tuberculosis.</a> Microorganisms. 8 (5), 701 (2020).</li><li>Lu, R., 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=SARS-CoV-2+detection+using+digital+PCR+for+COVID-19+diagnosis,+treatment+monitoring+and+criteria+for+discharge.">SARS-CoV-2 detection using digital PCR for COVID-19 diagnosis, treatment monitoring and criteria for discharge.</a> medRxiv. , (2020).</li><li>Nyaruaba, R., 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=Developing+multiplex+ddPCR+assays+for+SARS-CoV-2+detection+based+on+probe+mix+and+amplitude+based+multiplexing.">Developing multiplex ddPCR assays for SARS-CoV-2 detection based on probe mix and amplitude based multiplexing.</a> Expert Review of Molecular Diagnostics. , 1-11 (2020).</li></ol>

Few resources are available on how to develop RT-ddPCR assays for SARS-CoV-2 detection. Though not used in this article, standard samples with known copies may be used to develop and optimize assays. In this work however, SARS-CoV-2 samples grown in Vero-E6 cells were spiked in a background of human genomic RNA and used as standard samples to develop the assays. Proper primer and probe sequences are essential when developing assays. Since most preliminary work on SARS-CoV-2 RT-ddPCR used the China CDC primer and probes t...

Polymerase chain reaction (PCR), a well-recognized technique, has undergone several transformations since its advent to become a powerful technique capable of providing answers to nucleic acid research. These transformations have been a constant improvement of the old technique. These transformations can be summarized into three generations1. The first generation is conventional PCR that relies on gel electrophoresis to quantify and detect amplified targets. The second generation is quantitative real time PCR (qPCR) that can detect samples in real time and rely on a standard curve to directly quantify targets in a sample. The third generation, digital PCR (dPCR), can perform both detection, and absolute quantification of nucleic acid targets without the need of a standard curve. dPCR has also been improved further from reaction chambers being separated by the wells of a wall into emulsions of oil, water, and stabilizing chemicals within the same well as seen in droplet-based digital PCR2. In droplet digital PCR (ddPCR), a sample is partitioned into thousands of nanoliter-sized droplets containing individual targets that will later be quantified using Poisson statistics2,3,4. This technique gives ddPCR an edge in quantifying low abundant targets when compared to the other generations of PCR.
Recently, multiple applications have highlighted the superiority of ddPCR over the commonly used qPCR when detecting and quantifying low abundant targets1,5,6. SARS-CoV-2 is no exception to these applications7,8,9,10,11,12. Since the outbreak of SARS-CoV-2, scientists have been working on all fronts to come up with solutions on how to diagnose the virus and detect it efficiently. The current gold standard still remains to be qPCR13. However, RT-ddPCR has been shown to be more accurate in detecting low abundant SARS-CoV-2 targets from both environmental and clinical samples when compared to RT-qPCR7,8,9,10,11,12. Most of the SARS-CoV-2 ddPCR published works depend on simplex assays with the multiplex ones depending on commercial assays. Hence, more should be done to explain how to develop multiplex RT-dPCR assays for SARS-CoV-2 detection.
In a proper assay design, multiplexing can be used to save on cost, increase sample throughput, and maximize on the number of targets that can be sensitively detected within a small sample. When multiplexing with ddPCR, one must take account of how many fluorophores can be detected in a particular system. Some ddPCR platforms can support up to three channels while others support only two channels. Hence, when multiplexing with two channels, one has to use different approaches, including higher order multiplexing to detect more than two targets14,15,16. In this work, a two color ddPCR detection system is used to show steps on how to develop different SARS-CoV-2 RT-ddPCR assays that can be adapted for different research applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>32-channel fully automatic nucleic acid extractor Purifier 32</td>
			<td>Genfine Biotech</td>
			<td>FHT101-32</td>
			<td>Automated extractor for RNA</td>
		</tr>
		<tr>
			<td>AutoDG Oil for Probes</td>
			<td>BioRad</td>
			<td>12003017</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>ddPCR 96-Well Plates</td>
			<td>BioRad</td>
			<td>12003185</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Supermix for Probes (No dUTP)</td>
			<td>BioRad</td>
			<td>1863024</td>
			<td>Making ddPCR assay mastermix</td>
		</tr>
		<tr>
			<td>DG32 AutoDG Cartridges</td>
			<td>BioRad</td>
			<td>1864108</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>Electronic thermostatic water bath pot</td>
			<td>Beijing Changfeng Instrument and Meter Company</td>
			<td>XMTD-8000</td>
			<td>Heat inactivation of samples</td>
		</tr>
		<tr>
			<td>FineMag Rapid Bead Virus DNA/RNA Extraction Kit</td>
			<td>Genfine Biotech</td>
			<td>FMY502T5</td>
			<td>Magnetic bead extraction of inactivated RNA samples</td>
		</tr>
		<tr>
			<td>Pierceable Foil Heat Seals</td>
			<td>BioRad</td>
			<td>1814040</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet Tips for the AutoDG</td>
			<td>BioRad</td>
			<td>1864120</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>Pipet Tip Waste Bins for the AutoDG</td>
			<td>BioRad</td>
			<td>1864125</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>PrimeScript RT Master Mix (Perfect Real Time)</td>
			<td>TaKaRa</td>
			<td>RR036A</td>
			<td>cDNA generation</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer</td>
			<td>BioRad</td>
			<td>1814000</td>
			<td>Seal the droplet plate from AutoDG</td>
		</tr>
		<tr>
			<td>QuantaSoft 1.7 Software</td>
			<td>BioRad</td>
			<td>10026368</td>
			<td>Data acquisition and analysis</td>
		</tr>
		<tr>
			<td>QuantaSoft Analysis Pro 1.0</td>
			<td>BioRad</td>
			<td>N/A</td>
			<td>Data analysis</td>
		</tr>
		<tr>
			<td>QX200 Automated Droplet Gererator (AutoDG)</td>
			<td>BioRad</td>
			<td>1864101</td>
			<td>QX200 AutoDG consumable</td>
		</tr>
		<tr>
			<td>QX200 Droplet Reader</td>
			<td>BioRad</td>
			<td>1864003</td>
			<td>Droplet reading and data acquisition</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td>Droplet target amplification (PCR) and cDNA generation</td>
		</tr>
	</tbody>
</table>

two-step reverse transcription droplet digital pcr protocols for sars-cov-2 detection and quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

In a proof-of-concept study, the multiplex assays analytical performance was tested on clinical and research samples19. The performance of the multiplex assays was superior to that of an RT-PCR19. Since low numbers of droplets may indicate a problem during droplet generation, in this article a cutoff of 10,000 droplets per well was set based on empirical data.
A good separation between positive and negative droplets with minimal rain interference...

Watch this Scientific Journal Video about Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification at JoVE.com

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...

These protocols can be used to develop in-house multiplex ddPCR assays for detection of SARS-CoV-2 using a two-color ddPCR system. This is useful for people who want to develop their own kits for the detection of SARS-CoV-2 and other pathogens. Compared to the gold standard RT-qPCR, RT-ddPCR can be used to quantify multiple SARS-CoV-2 genes without the need of a standard curve.Since ddPCR is an emerging technique, visual demonstration is important to help current and future users understand and easily develop multiplex assays for SARS-CoV-2 detection and other pathogens. For cDNA synthesis, add two microliters of 5X reverse transcription master mix, five microliters of the RNA sample of interest, and three microliters of RNase-free double distilled water into a 100 or 200 microliter PCR tube to a final volume of 10 microliters as solution, and place the tube on ice. When off the reagents have been added, load the sample into a thermal cycler and set the instrument to run a reverse transcription at 37 degrees Celsius for 15 minutes, a heat inactivation of the reverse transcription for 5 seconds at 85 degrees Celsius, and a final infinite hold at four degrees Celsius.Before performing droplet digital PCR, add 19.8 microliters of freshly prepared master mix to the appropriate number of wells of a nuclease free 96-well droplet digital PCR plate. Add 2.2 microliters of cDNA to each well of master mix to a final volume of 22 microliters of reaction mix per well and seal the plate with a disposable PCR plate sealer. Then, vortex the plate for 15 to 30 seconds in centrifuge for 10 to 15 seconds to collect the well contents at the bottom of the plate.For automated droplet generation on the automated droplet generator touchscreen, click configure sample plate and select the columns in which the samples are located on sample plate. Click okay and open the automated droplet generator door. Load the sample and droplet generation plates and the other materials necessary for the experiment into the respective locations within the instrument and close the door.When the touchscreen turns green, indicating that all of the materials have been loaded into their correct positions, select oil for probes and start droplet generation. Wait for the droplet generation to run to completion according to the time indicated on the screen. When the screen says Droplets ready, open the door to remove the plate containing the droplets and use a plate sealer for five seconds at 180 degrees Celsius to seal the plate with a pierceable foil seal.Within 30 minutes of the droplet generation, place the sealed droplet plate into a 96 deep well thermal cycler. Set the sample volume to 40 microliters and the lid temperature to 105 degrees Celsius. Then, PCR amplify the droplets.For droplet reading, after the amplification, transfer the thermal cycled 96-well plate into a droplet reader and open the accompanying software on a computer connected to the droplet reader. From the setup mode, select New and double click on any well to open the well editor dialogue box. Select the wells to be read and set experiment to absolute quantification, supermix to droplet digital PCR supermix for probes.Target one type to channel 1 unknown and target two type to channel 2 unknown. Assign the sample names based on the plate layout and click apply and okay. After saving the template, click Run.When prompted by the software, select the FAM/HEX color. At the end of data acquisition, remove the plate from the reader. Before analyzing the data, check the data from all of the wells to obtain the total number of droplets.And set a cutoff to accept a minimum number of droplets to be counted as positive or negative results. For simplex and duplex assay analysis, double click on the QLP file to be analyzed and select Analyze. Use the 1D amplitude and 2D amplitude threshold tools to distinguish between the positive and negative droplets for each well in the correct channel, using the node template control and positive control samples as a guide.Then, export the results as a CSV file for additional analysis. For a triplex probe mix assay, open the QLP file and select the wells to be analyzed in the plate editor tab. Set the experiment type to direct quantification and the assay to probe mix triplex, and enter the target name.Then click Apply. Use the graph tools to assign specific colors to different target clusters as directed by the select to assign cluster window popup suggestions. When all of the target colors have been assigned, the quantification data will be displayed in the well data window.Export the data as a CSV file for downstream analysis. For a quadruplex assay, set the experiment type to direct quantification and the assay to amplitude multiplex. Enter the target name and click Apply.Use the graph tools to assign specific colors to different target clusters as directed by the select to assign cluster window popup suggestions. When all of the target colors have been assigned, the quantification data will be displayed in the well data window. Export the data as a CSV file for additional analysis.As observed in this representative temperature gradient analysis, higher kneeling temperatures could not clearly distinguish positive droplets from negative droplets when a duplex assay was run. However, with a decrease in a kneeling temperature, an optimal separation between positive and negative droplets was achieved. Using a two color RT droplet digital PCR detection system, it is possible to detect one, two, three, and four SARS-CoV-2 targets within a single sample.A no template control sample can be used to locate negative droplets to help set the negative threshold in simplex assays. In duplex assays, the analysis can be performed in individual channels or in the 2D amplitude. Although higher order multiplex assays data analysis is not straightforward, these assays are useful, because they allow up to eight droplet clusters to be assessed in triplex probe mix assays and up to 16 droplet clusters for quadruplex amplitude-based assays.Depending on the assay to be developed, make sure the correct target primer and probe concentrations are added to the master mix. Using this procedure, one may alternate the targets to develop novel RT-ddPCR assays. This assays can be used for different research purposes or for the diagnosis of emerging infectious diseases.Since its development, this technique has been used for the accurate determination of viral nucleic acid copies in different standards, the human body, and environmental samples.

two-step-reverse-transcription-droplet-digital-pcr-protocols-for-sars

Research

JoVE Journal

Biology

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...

Improved Protein Quantification Using Two-Step Mitochondrial Isolation

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Author Spotlight: Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial ...

Quantification of Viral Vectors in Tears Using ddPCR for Bioshedding Studies

A Validatable Droplet Digital Polymerase Chain Reaction Assay for the Detection of Adeno-Associated Viral Vectors in Bioshedding Studies of Tears

The use of viral vectors to treat genetic diseases has increased substantially in recent years, with over 2,000 studies registered to date. Adeno-associated viral (AAV) vectors have found particular success in the treatment of eye related diseases, as exemplified by the approval of voretigene neparvovec-rzyl. To bring new therapies to market, regulatory agencies typically request qualified or validated bioshedding studies to evaluate release of the vector into the environment. However, no official guidelines for the development of molecular based assays to support such shedding studies have been released by the United States Food and Drug Administration, leaving developers to determine best practices for themselves. The purpose of this protocol is to present a validatable protocol for the detection of AAV vectors in human tears by droplet digital polymerase chain reaction (ddPCR) in support of clinical bioshedding studies. This manuscript discusses current industry approaches to molecular assay validation and demonstrates that the method exceeds the target assay acceptance criteria currently proposed in white papers. Finally, steps critical in the performance of any ddPCR assay, regardless of application, are discussed.

Author Spotlight: Addressing Regulatory Gaps in Molecular Studies by Quantifying Viral Vectors in Complex Matrices 

Here, we present a protocol for the development and validation of good laboratory practices in the compliant detection of adeno-associated viral vectors in human tears by droplet digital polymerase chain reaction in support of clinical development of gene therapy vectors.