Name: high-throughput quantitative real-time rt-pcr assay for determining expression profiles of types i and iii interferon subtypes
Uploaded: 2015-03-24T16:00:00
Duration: 10 min
Description: Watch this Scientific Journal Video about High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes at JoVE.com

This work was supported by CBER/FDA-NIAID/NIH Interagency Agreement YI-AI-6153-01, FDA/CBER intramural funds, and the FDA Medical Countermeasures Initiative. TCT, MNB, VPM, LMS, and KDK were supported by an appointment to the Research Participation Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute for Science Education through an Interagency agreement between the U.S. Departmen of Energy and the U.S. Food and Drug Administration.

1. Preparation of Standard Serial Dilutions (Figure 3A)
NOTE: Serial dilution series of linearized plasmids containing the sequences targeted by a Pr/Pb set are used as quantitative standards for the qRT-PCR assay. Each standard serial dilution set for the IFN subtypes contains enough volume to run 90 assay plates. The four points of the standard dilution curve used for the assay are selected to cover Ct values from a range of 20 to 32 (Table 3).
<ol>
	<li>Thaw, vortex, and briefly centrifuge the standard (50 pM) and salmon sperm DNA (SSDNA, 10 mg/ml) stock solutions.</li>
	<li>Prepare the SSDNA/water mix for the 17 standard dilution sets by mixing 51 &#181;l of SSDNA with 20.3 ml of water.</li>
	<li>Label one 8-tube PCR strip for a standard dilution set. 
	NOTE: Preparation of the standard serial dilutions from the stock solutions will take 2-3 hr.</li>
	<li>Dispense 190 &#181;l of the SSDNA/water mix to the first tube in the strip and 180 &#181;l of SSDNA/water mix to the five remaining tubes.</li>
	<li>Perform a 1:20 dilution by transferring 10 &#181;l from the 50 pM standard stock to the tube with 190 &#181;l of the SSDNA/water, mix; vortex and quickly centrifuge the PCR strip.</li>
	<li>Perform a 1:10 dilution by transferring 20 &#181;l from the most recently diluted tube to the next tube in the series; vortex and quickly centrifuge the PCR strip.</li>
	<li>Repeat step 1.6 until the last tube in the dilution series has received the standard.</li>
	<li>Repeat steps 1.3-1.7 for each standard.</li>
</ol>
See Table 3 Below
2. Preparation of Primer/Probe (Pr/Pb) and No Template Control (NTC) Working Stock Mixes (Figure 3B)
NOTE: Each 1.7 ml Pr/Pb set working stock and 128.6 &#181;l No Template Control (NTC) working stock mix will make 30 assay plates.
<ol>
	<li>Prepare the Pr/Pb set working stock mixes. 
	NOTE: Preparation of the Pr/Pb set and NTC working stock mixes from the stock solutions will take 2-3 hr.
	<ol>
		<li>Resuspend the primers and probes at 100 &#181;M with ultra-pure water for preparation of stock solutions.</li>
		<li>Label up to seventeen 2 ml tubes; one for each Pr/Pb set included in the assay.</li>
		<li>Thaw, vortex, and briefly centrifuge the primers (100 &#181;M), probes (100 &#181;M), and SSDNA (10 mg/ml) stock solutions.</li>
		<li>Add 2 &#181;l of SSDNA to every tube using the 12.5 &#181;l electronic multichannel pipette. Add the appropriate forward primer, reverse primer, and probe to each Pr/Pb set working stock tube. See Table 1 for the Pr/Pb sets targeting human IFN subtypes and Table 2 for the rhesus macaque Pr/Pb sets. Add water to bring the volume of each Pr/Pb set working stock tube to 200 &#181;l. 
		NOTE: The volume of primers, probe, and water to add depends on the required final reaction concentration of a Pr/Pb set.</li>
	</ol>
	</li>
	<li>Prepare the NTC working stock mixes.
	<ol>
		<li>Label two 5-tube PCR strips for the NTC working stocks mixes.</li>
		<li>Transfer 14.3 &#181;l of each Pr/Pb set to the appropriate NTC working stock mix tube. See Table 4 for the Pr/Pb set combinations for NTC wells. Add water to each of the NTC working stock mixes to bring the final volume to 128.6 &#181;l.</li>
		<li>Vortex, briefly centrifuge, and place the tubes in the dark on ice or at -20 &#176;C for long term storage.</li>
	</ol>
	</li>
</ol>
See Table 4 Below
<ol start="3">
	<li>Dilute the Pr/Pb set working stocks.
	<ol>
		<li>Following the removal of an aliquot of the Pr/Pb sets required for the NTC working stock mixes, add 1.5 ml of water to bring the final volume of each Pr/Pb set working stock tube to 1.7 ml.</li>
		<li>Vortex, briefly centrifuge, and place the tubes in the dark on ice or at -20 &#176;C for longer term storage.</li>
	</ol>
	</li>
</ol>
3. Preparation of 384-well assay plates using the automated multichannel pipettor
<ol>
	<li>Prepare a 96-well source plate of the Pr/Pb sets and NTC mixes for aliquoting to 384-well assay plates. 
	NOTE: Preparation of the source plate from the Pr/Pb workings stock mixes will take less than 1 hr. Each 96-well source plate is enough to make six 384-well assay plates (Figure 3C).
	<ol>
		<li>Vortex and briefly centrifuge the Pr/Pb set working stocks and NTC working stock mixes.</li>
		<li>Place a new 96-well plate in a chilled 96-well cooling block and designate the wells for each Pr/Pb set or NTC mix the wells receive (see Figure 2).</li>
		<li>Add water to the 96-well plate using a 250 &#181;l electronic multichannel pipette: Dispense 66 &#181;l of water to every Pr/Pb set well (except the 4 wells for Target 17). Dispense 82.5 &#181;l of water to the 4 Target 17 wells. Dispense 27.5 &#181;l of water to every NTC mix well.</li>
		<li>Add the correct Pr/Pb set working stock to the designated wells of the 96-well plate: Dispense 54 &#181;l of the correct Pr/Pb set working stock to the designated Pr/Pb set wells (except the 4 wells for Target 17). Dispense 67.5 &#181;l of the Target 17 Pr/Pb set working stock to the designated Target 17 Pr/Pb set wells. Add 22.5 &#181;l of the correct NTC working stock mix to the designated wells.</li>
		<li>Seal the 96-well plate with an adhesive plate seal and centrifuge for 1 min at 700 x g to ensure the contents are at the bottom of the wells.</li>
		<li>Place the 96-well plate in the vortex mixer using a 96-well plate adaptor and mix for 1 min at 1,000 rpm. Centrifuge the 96-well plate for 5 min at 700 x g.</li>
		<li>Store the 96-well source plate in the dark at 4 &#176;C if using in the same day, otherwise store at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Prepare the 384-well assay plates by adding the Pr/Pb sets using the automated multichannel pipettor. 
	NOTE: Preparation of six assay plates from the 96-well source plate will take 3-4 hr.
	<ol>
		<li>Prior to making plates, pre-chill the cooling nest to 4 &#176;C and pre-heat the plate evaporator to 125 &#176;C and the bottom heat block to 80 &#176;C with airflow blowing between 20-25 liters&#160;per min (LPM).</li>
		<li>Switch on the automated multichannel pipettor and open the protocol for making IFN assay plates by double clicking the software icon.</li>
		<li>To setup the platform, place a completely full pipet tip box in platform position 1, the 96-well source plate in position 4, and a new 384-well plate in position 6. Begin the run by pressing the play button. 
		NOTE: Upon completion of a run, each well will contain 5 &#181;l of Pr/Pb mix. 
		NOTE: The final amount of SSDNA added per well of the 384-well assay plate is 0.025 &#181;g.</li>
		<li>Gently tap the 384-well assay plate on a flat surface to ensure fluids are at the bottom of the wells and apply an adhesive plate seal.</li>
		<li>Centrifuge the plate for 1 min at 700 x g. Remove the adhesive plate seal, place the 384-well assay plate into the plate dryer and position the 384-well manifold directly above the wells.</li>
		<li>When the 384-well assay plate&#8217;s contents dry, apply a new adhesive plate seal, wrap in foil, label, and store in the dark at 4 &#176;C until use. 
		NOTE: Plates can be stored at 4 &#176;C for at least 6 months.</li>
		<li>Repeat steps 3.2.3-3.2.6 until 6 x 384-well assay plates are prepared or the liquid in the 96-well source plate is depleted.</li>
		<li>Turn off the cooling nest, close the software, and switch off the automated multichannel pipettor. Turn off the plate dryer.</li>
	</ol>
	</li>
</ol>
4. Loading and Running a 384-well Assay Plate
<ol>
	<li>Prepare two housekeeping gene (HKG) well mixes, which consist of the Pr/Pb set for a HKG, SSDNA, PCR master mix, and water, to be added to a dried 384-well assay plate (Figure 3D). 
	NOTE: Preparation of the mixes and loading the assay plate will take 1-2 hr. Running the assay plate will take less than 2 hr.
	<ol>
		<li>Label a 1.5 ml tube for each HKG mix.</li>
		<li>Dilute 2 &#181;l of SSDNA (10 mg/ml) with 84.9 &#181;l water. Add 2 &#181;l of the diluted SSDNA, 11.8 &#181;l of water, 23 &#181;l of master mix, and 9.2 &#181;l of the correct 20x HKG Pr/Pr set to each tube, vortex, and briefly centrifuge.</li>
		<li>Use glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ubiquitin C (UBC) as the HKG for the human assay (see Materials Table). Use GAPDH and 18S ribosomal RNA as the HKG for the rhesus assay. 
		NOTE: The HKG used to perform the assay are flexible and should reflect the genes appropriate for the cell type being tested. GAPDH, UBC, and 18S are examples of commonly used HKG; other HKG may be more appropriate.</li>
	</ol>
	</li>
	<li>Prepare the sample and positive control well mixes, which consist of the sample or positive control cDNA, master mix, and water, to be added to a dried 384-well assay plate. Prepare the sample and the positive control mixes in enough quantity to dispense to 21 plate wells (Figure 3D).
	<ol>
		<li>Prior to cDNA synthesis, follow the manufacturer&#8217;s instructions to prepare the RNA samples with a DNase digestion step.</li>
		<li>Prepare the samples and positive control in advance from the cDNA synthesis of at least 500 ng of RNA followed by treatment with RNase H (final volume of 24 &#181;l for each sample) following the manufacturer&#8217;s instructions. Store the prepared cDNA at -20 &#176;C until use. Thaw, vortex, and briefly centrifuge the sample cDNA.</li>
		<li>Add 78.8 &#181;l of master mix and 54.8 &#181;l of water to each 24 &#181;l sample and positive control tube.</li>
		<li>Vortex and briefly centrifuge the tubes.</li>
	</ol>
	</li>
	<li>Prepare the standards and NTC well mixes, consisting of master mix and water, to be added to a dried 384-well assay plate (Figure 3D). For the standards well mix, add 165 &#181;l of water to 275 &#181;l of master mix in a 1.5 ml tube. For the NTC well mix, add 52.5 &#181;l of water to 52.5 &#181;l of master mix in a 1.5 ml tube. Vortex and briefly centrifuge the tubes.</li>
	<li>Prepare a dried 384-well assay plate for loading of the mixes and samples.
	<ol>
		<li>Remove a dried 384-well assay plate from 4 &#176;C and centrifuge for 1 min at 700 x g.</li>
		<li>Place the plate on a chilled 384-well cooling block, remove the adhesive plate seal and outline the wells of the plate to designate where the various mixes and samples will be pipetted (Figure 1).</li>
	</ol>
	</li>
	<li>Load the 384-well assay plate with standard well mix, standards, NTC well mix, samples, positive control, and HKG mix (Figure 3E). Vortex and briefly centrifuge all solutions before dispensing to the plate.
	<ol>
		<li>Dispense 6 &#181;l of the standards well mix to each standard well with the 30 &#181;l electronic multichannel pipette. Dispense 1.5 &#181;l of the correct standard serial dilution to the designated well with the 12.5 &#181;l electronic multichannel pipette.</li>
		<li>Dispense 7.5 &#181;l of NTC well mix to each NTC well with the 30 &#181;l electronic multichannel pipette. Dispense 7.5 &#181;l of the sample to the designated wells with the 30 &#181;l electronic multichannel pipette. Dispense 2.5 &#181;l of the HKG Pr/Pb mixes to the designated wells with the 12.5 &#181;l electronic multichannel pipette.</li>
		<li>Fill any empty wells with 7.5 &#181;l of the leftover water and master mix mixture with the 30 &#181;l electronic multichannel pipette; 7.5 &#181;l of water alone will also work.</li>
		<li>Seal the 384-well assay plate with the optical adhesive film and centrifuge for 1 min at 700 x g. Vortex the sealed 384-well plate in the vortex mixer for 2 min at 2,600 rpm and centrifuge for 5 min at 700 x g.</li>
		<li>Place the sealed 384-well assay plate in the qRT-PCR machine, open the template for the assay layout and begin the run. 
		NOTE: Results can be exported as a spreadsheet or as a text file for analysis.</li>
		<li>Use the following optimal thermal cycler reaction conditions for the assay: i) 50 &#176;C for 2 min, ii) 95 &#176;C for 10 min, iii) 95 &#176;C for 25 s, iv) 59 &#176;C for 1 min. Repeat steps iii and iv for 40 cycles.</li>
		<li>Export the raw data from the qRT-PCR platform into a spreadsheet application software. Plot each 4 point standard set as a standard curve to observe linearity. Perform analysis by calculating the &#8710;Cq or by copy number based on the four point standard curves27.</li>
	</ol>
	</li>
</ol>
See Figure 3 Below

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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=High-throughput+quantitative+real-time+polymerase+chain+reaction+array+for+absolute+and+relative+quantification+of+rhesus+macaque+types+I,+II,+and+III+interferon+and+their+subtypes.">High-throughput quantitative real-time polymerase chain reaction array for absolute and relative quantification of rhesus macaque types I, II, and III interferon and their subtypes.</a> Journal of interferon &amp; cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 32, 407-415 (2012).</li><li>Wang, Q., Chen, L., Long, Y., Tian, H., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+beacons+of+xeno-nucleic+acid+for+detecting+nucleic+acid.">Molecular beacons of xeno-nucleic acid for detecting nucleic acid.</a> Theranostics. 3, 395-408 (2013).</li><li>Little, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification-refractory+mutation+system+(ARMS)+analysis+of+point+mutations.">Amplification-refractory mutation system (ARMS) analysis of point mutations.</a> Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.]. 9 (Unit 9 8), (2001).</li></ol>

VPM and RLR are co-inventors of the technology to measure expression of IFN- and IFN- subtypes and derive royalty income from it. The remaining authors declare no conflict of interest.

This report describes design, batch production, and an approach towards analysis of an assay to measure transcription of a set of highly similar genes in a research laboratory setting. The high-throughput qRT-PCR assay reported here measures the IFN- and -&#955; subtypes with high specificity. This method involves two key aspects, the design of Pr/Pb sets that discriminate between members of a homologous gene family and the development of a production platform for the creation of reliable and consistent 384-well assay plates preloaded with the Pr/Pb sets. The qRT-PCR probes incorporate a structural (MB) or chemical (LNA) approach towards enhancing their specificity29. For two of the primer sets in the rhesus assay (Table 2), the amplification-refractory mutation system (ARMS) was incorporated into the primer sequences to further enhance specificity30. While it is generally best to target exon-exon junctions to enhance specificity of a qRT-PCR reaction, this was not possible because the type I IFN genes lack introns. Therefore, genomic DNA will be amplified in the PCR reaction, and must be degraded by DNase treatment after RNA extraction.
The target region for the Pr/Pb sets was restricted to the coding regions of the mature peptide for each IFN. Because of the high sequence similarity among the IFN-&#945; subtypes, particularly the mature peptide region, it was sometimes necessary to compromise sensitivity to ensure specificity. This was particularly the case with IFN-&#945;17, where the mature peptide transcript has only four unique bases when compared against the other IFN-&#945; subtypes. Targeting IFN-&#945;17 required primers that bind the transcripts of multiple subtypes, restricting specificity to the probe. As a consequence, the PCR reaction will amplify subtypes other than IFN-&#945;17, thereby consuming a substantial percentage of the PCR reagents and lowering the amplitude of the fluorescence signal from the specific probe for IFN-&#945;17. An additional challenge towards designing sensitive and specific Pr/Pb sets for highly similar genes such as the IFN is the possibility that the annotated sequences in the GenBank database may not be complete or comprehensive at time of design. Again, this was a challenge for IFN-&#945;17, in which newly annotated sequences do not perfectly align with the version of the sequence in the database at the time of design. Therefore, when designing Pr/Pb sets for genes that have not been intensively studied, it is wise to periodically check the latest annotated sequence of a target gene. Finally, it is necessary to ensure that the Pr/Pb sets do not amplify pseudogenes that may be transcribed but not translated.
After designing the Pr/Pb sets, the next challenge is optimizing the PCR conditions of seventeen different Pr/Pb sets on one 384-well plate. Transcript standards are important in testing the specificity of a Pr/Pb set and become essential for harmonizing the qRT-PCR conditions of the numerous different PCR reactions. Testing a Pr/Pb set against the transcript standards establishes its efficiency and sensitivity; plasmids that express highly similar pseudogenes may be necessary to ensure that the Pr/Pb set selectively measures transcription of the functional gene. The transcript standards also provide a quantitative means of analysis (number of transcripts), in addition to the semi-quantitative analysis relative to a housekeeping gene (&#916;Cq).
Robotic multichannel pipetting from a 96-well source plate into multiple 384-well assay plates improves the precision and consistency of inter-plate results. Salmon sperm DNA (SSDNA) is used as a carrier that stabilizes and preserves the Pr/Pb sets for long term storage, as does drying the reagents dispensed into the plates. Drying the plates also decreases the volume of the reaction necessary for reproducible results, which in turn preserves precious samples and decreases the use of costly reagents. Through these steps, batches of plates are assembled that provide precision and consistency for more than six months.
Following plate preparation, quality control measures are critical to check the consistency of a batch of plates. For this purpose, an additional four sets of standards are run on a plate. The &#8220;5x standard&#8221; plate tracks performance and creates a data set to which an individual plate&#8217;s standards are compared. While ten 10-fold dilutions of each standard are used during assay design, space considerations require that four points are used for the standard curve on each assay plate, and for the 5x standard plate. Additionally, a positive control should be included on each plate to ensure the validity of the plate.
Typically, it takes 3-4 hr to prepare six assay plates from one Pr/Pb source plate; it is feasible to prepare twelve plates in a single day in a research laboratory. Since each human IFN subtype assay plate examines seventeen Pr/Pb sets and can accommodate fifteen experimental samples, one day of assembly produces a batch of plates with the capacity to generate up to 3,060 experimental data points. Raw data from the qRT-PCR platform can be processed and assembled using programming scripts in a spreadsheet application software to automatically fill a predesigned analysis template. This method minimizes hands-on data entry, thereby preventing copying errors and allows the investigator to focus on data analysis rather than data assembly. As described here, this high-throughput qRT-PCR assay can be applied to measure the expression of interferon subtypes in human or rhesus macaque samples and could be adapted to use for other species or homologous gene sets. The flexibility of the plate layout allows the user to change primer/probe sets to tailor the genes of interest toward a particular cell type or model system. This assay can be applied to measure IFN expression signatures in cell culture models studying pathogens or in patient samples in the context of disease models to elucidate the signaling mechanisms involved in immune response.

Types I and III interferons (IFN) are critical in the immune response to viruses and other pathogenic stimuli and present in all vertebrates1. Immune and non-immune cells express and secrete, as well as respond to, IFN2. Innate immune sensors, such as toll-like receptors (TLR), STING, and RIG-I, induce type I and III IFN expression upon detection of pathogen associated molecular patterns (PAMP)3,4. In humans, type I IFN include IFN-&#946;, -&#969;, -&#954;, and 12 subtypes of IFN-&#945;, and bind to the IFNAR1/IFNAR2 receptor complex2,5. Type III IFN include IFN-&#955;1, -&#955;2, and -&#955;3 and bind to the IL10RB/IL28RA receptor complex2. Classically, types I and III IFN bind to their respective receptor complexes which then recruit STAT1/STAT2 heterodimers and initiate transcription of interferon stimulated genes (ISG)6. ISG are involved in a diverse range of functions, from antiviral and antiproliferative activity to activation of the adaptive immune response7.
The numerous mechanisms pathogens have evolved to evade, subvert, and hijack elements of the IFN signaling pathway demonstrate the importance of IFN in the innate immune response8. For example, vaccinia virus expresses a decoy receptor with IFNAR1 homology that sequesters type I IFN9 while a Yaba-like disease virus secretes a glycoprotein that inhibits type I and III IFN proteins10. In addition to their role in host defense, IFN are also implicated in cancer surveillance and a number of auto-inflammatory diseases: silencing of IFN expression in breast cancer cells restricts immunosurveillance11, overproduction of IFN-&#945; is a mechanism in the development of systemic lupus erythematosus12, and errant activation of STING leads to systemic inflammation caused by excessive amounts of IFN in STING-associated vasculopathy13. Therapeutically, IFN are used to treat multiple sclerosis14, chronic viral infections such as HBV15 and HCV16,17, and cancers such as hairy cell leukemia18 and chronic myelogenous leukemia19. Questions about the relevance of IFN in a particular physiological process continuously reveal the ubiquitous nature of this cytokine family.
Type I IFN, especially the IFN-&#945; subtypes, are often considered as one entity20-23, rather than as a group of closely related, but distinct, proteins. The existence and persistence of multiple IFN species including the IFN-&#945; subtypes, throughout vertebrate evolution24 suggests that at least a subset of these subtypes have specific or unique functions. It is possible that defining patterns of IFN expression will decipher and help characterize the specific functions of one or more of the subtypes17. The challenge of studying the type I and III IFN subtypes is based on their shared sequence identity: the twelve IFN-&#945; subtypes share &#62;50%25 and the IFN-&#955; subtypes share 81-96%26 of their amino acid sequences. In the described qRT-PCR assay, molecular beacon (MB) and locked nucleic acid (LNA) fluorescent probes discriminate single base pair differences between the highly similar IFN subtype sequences and allow for the characterization of the IFN expression signature. The assay&#8217;s 384-well plate format includes both quantitative (transcript standards) and semi-quantitative (the housekeeping genes (HKG)) measures, allowing for analysis by transcript copy number and &#8710;Cq respectively. Batch assembly, facilitated by automated multichannel pipetting, and long term storage, possible through drying the primer/probe (Pr/Pb) sets on the plates, enhance the reproducibility, utility, and practicality of the assay.
This protocol describes the process for preparing 384-well qRT-PCR assay plates (Figure 1) with up to seventeen different Pr/Pb sets targeting human IFN subtypes (Table 1). Pr/Pb set working stock source plates (Figure 2) are used to prepare multiple 384-well assay plates in a process that can be automated using a robotic multichannel pipettor. While the initial focus was on creating a protocol for studying human IFN expression signatures, this method has been applied to rhesus macaques as well. Though the plate layouts are slightly different and the Pr/Pb sets (Table 2) are distinct, the overall preparation method for creating the human and rhesus plates is identical. With minimal modifications of the protocol, the method could be executed to allow for the development of assays to study other groups of closely related genes.
See Figures 1 and 2 Below 
See Tables 1 and 2 Below

<table border="0" cellpadding="0" cellspacing="0" width="1824">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;"></td>
			<td style="width:668px;"></td>
			<td style="width:141px;"></td>
			<td style="width:531px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2mL PCR Tube Strips&#160;</td>
			<td>Eppendorf (Westbury, NY, USA)</td>
			<td>E0030 124 286</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-channel 384 Equalizer Electronic Pipette, 0.5-12.5uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-139</td>
			<td>*Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-channel 384 Equalizer Electronic Pipette, 1-30uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-137</td>
			<td>*Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">12-channel Electronic Pipette, 5-250uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td style="width:141px;">14-387-118</td>
			<td>*Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.0mL Micro Centrifuge tube, sterile</td>
			<td>Celltreat Scientific Products (Shirley, MA, USA)</td>
			<td>229446</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">8-channel Electronic Pipette, 15-1250uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-115</td>
			<td>*Discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable Reagent Reservoirs&#160;</td>
			<td>VWR International (Radnor, PA, USA)</td>
			<td>89094-668</td>
			<td>25mL reservoirs with 2 dividers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E-Centrifuge</td>
			<td>Wealtec Corp. (Sparks, NV, USA)</td>
			<td>1090003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eukaryotic 18S rRNA (18S)</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>Hs99999901_s1</td>
			<td>Rhesus HKG Primer/probe set</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fixed Speed Mini Vortexer</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>02-215-360</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gas Permeable Adhesive Plate Seal</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>AB-0580</td>
			<td>Disposable plate seal used during the plate preparation process&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)</td>
			<td>FBR (Silver Spring, MD, USA)</td>
			<td>Custom Order</td>
			<td>Human HKG Primer/probe set</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>Rh02621745_g1</td>
			<td>Rhesus HKG Primer/probe set</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hudson Solo automated multichannel pipettor&#160;</td>
			<td>Hudson Robotics (Springfield, NJ, USA)</td>
			<td>800200</td>
			<td>Modified with a 384-well cooling nest</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Linearized plasmids (IFN genes)</td>
			<td>Aldevron (Fargo, ND, USA)</td>
			<td>Custom Order</td>
			<td>Item 3000, 3580; used for assay standards&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LNA inhibitors</td>
			<td>Exiqon (Woburn, MA, USA)</td>
			<td>Custom Order</td>
			<td>Item 500100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LNA Probes</td>
			<td>Sigma-Proligo (St. Louis, MO, USA)</td>
			<td>Custom Order</td>
			<td>Material number VC00023</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix Filtered Pipet Tips, 12.5uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-193</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix Filtered Pipet Tips, 30uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-196</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix TallTip Extended Length Pipet Tips, 1250uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix TallTip Extended Length Pipet Tips, 250uL</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>14-387-152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Optical 384-Well Reaction Plate with Barcode</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>4309849</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Optical 96-Well Reaction Plate</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>N8010560</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Optical Adhesive Film</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>4311971</td>
			<td>Adhesive film used for sealing the plate prior to the qRT-PCR run&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsoft Excel 2010 Spreadsheet</td>
			<td>Microsoft (Redmond, WA, USA)&#160;</td>
			<td>Office 2010</td>
			<td>Visual Basic programming language&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MixMate Vortex Mixer</td>
			<td>Eppendorf (Westbury, NY, USA)</td>
			<td>5353 000.014</td>
			<td>2 dimensional plate vortex apparatus</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Beacon Probes</td>
			<td>FBR (Silver Spring, MD, USA)</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR Cooler, iceless cold storage system for 96 well plates and PCR tubes</td>
			<td>Eppendorf (Westbury, NY, USA)</td>
			<td>Z606634-1EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plate Dryer (manifold)</td>
			<td>Mechanical and Electrical Design Fabrications, NIH Office of Research Services (Bethesda, MD, USA)</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primer sets</td>
			<td>FBR (Silver Spring, MD, USA)</td>
			<td>Custom Order</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pUC57 plasmid DNA</td>
			<td>Genescript (Piscataway, NJ, USA)</td>
			<td>SD1176</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rnase-Free 1.5mL Microfuge Tubes</td>
			<td>Life Technologies (Grand Island, NY, USA)</td>
			<td>AM12400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-free DNase Set (50)</td>
			<td>Qiagen (Valencia, CA, USA)</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase H</td>
			<td>New England Biolabs (Ipswitch, MA, USA)</td>
			<td>M0297L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit (250)</td>
			<td>Qiagen (Valencia, CA, USA)</td>
			<td>74106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Robot Tips (clear tip pre-sterilized pipet tips)</td>
			<td>Hudson Robotics (Springfield, NJ, USA)</td>
			<td>800350-S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Salmon Sperm DNA Solution</td>
			<td>Invitrogen (Carlsbad, CA, USA)</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SoftLinx Version V</td>
			<td>Hudson Robotics (Springfield, NJ, USA)</td>
			<td>Custom Order</td>
			<td>Software for programming pipetting steps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Fast Universal PCR Master Mix (2x), no AmpErase UNG</td>
			<td>Life Technologies (Grand Island, NY, USA)</td>
			<td>4352042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ABgene Adhesive Plate Seals&#160;&#160;</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>AB0580</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:485px;">Ubiquitin C (UBC)</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>Hs01871556_s1</td>
			<td>Human HKG Primer/probe set</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Verso cDNA synthesis Kit</td>
			<td>Thermo-Fisher Scientific (Waltham, MA, USA)</td>
			<td>AB1453B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ViiA 7 Real-Time PCR System</td>
			<td>Applied Biosystems (Foster City, CA, USA)</td>
			<td>4453536</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water-Ultra Pure</td>
			<td>Quality Biological, INC. (Gaithersburg, MD, USA)</td>
			<td>351-029-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zipvap 384 (plate dryer heating element)</td>
			<td>Glas-Col LLC (Terre Haute, IN, USA)</td>
			<td>384-109A</td>
			<td>Plate Dryer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput quantitative real-time rt-pcr assay for determining expression profiles of types i and iii interferon subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

The qRT-PCR assay described can be implemented to analyze expression patterns of types I and III IFN in a variety of cell types and contexts. For example, human type I and III IFN expression signatures were analyzed in peripheral blood mononuclear cells (PBMC) from 6 donors stimulated with TLR ligands; poly I:C (25 &#956;g/ml), LPS (10 ng/ml), imiquimod (10 &#956;M), and CpG oligonucleotides (1 &#956;M) (Figure 4). Data were analyzed using a spreadsheet application software and presented as radar charts with the IFN-&#945; subtypes arranged clockwise according to the phylogenetic tree of their protein sequence27. The radar charts of human IFN expression are presented in a log scale calculated using the two different methods of analysis incorporated into the assay design: absolute Cq value normalized to HKG (&#916;Cq), and copy number of template per microgram (&#956;g) of total RNA. Copy number values are calculated from the results of a transcript&#8217;s standard curve. The data shows that human IFN expression signatures elicited by TLR agonists are ligand specific.
See Figure 4 Below
As demonstrated for the human IFN expression signatures, expression signatures of types I and III IFN in rhesus macaques were also TLR ligand specific. PBMC from 3 donors were stimulated with poly I:C (50 &#956;g/ml), LPS (10 &#956;g/ml), and imiquimod (10 &#956;g/ml) for 3 hr (Figure 5). IFN subtype expression in unstimulated cells at baseline was low. A limited number of IFN- subtypes were expressed in response to LPS and poly I:C. In contrast, IFN expression in response to imiquimod was high and the subtype expression was broad. Expression of IFN-&#946; and IFN-&#955;1 was enhanced by all three TLR agonists28.
See Figure 5 Below
<img alt="Figure 1" src="/files/ftp_upload/52650/52650fig1.jpg" /> 
Figure 1: The layout for a 384-well assay plate with seventeen Pr/Pb sets. Target number refers to a Pr/Pb set. The four point standard curves (dark grey triangle) are added to columns 1-5. The HKG Pr/Pb sets (white background) are added to columns 6 and 7. The remaining columns, 8-24, are specific for one of the seventeen Pr/Pb sets. Samples are added to rows A-P, from columns 6-24 (black arrows). The two wells (O5, P5) at the bottom of column 5 receive only water and master mix. <a href="https://www.jove.com/files/ftp_upload/52650/52650fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52650/52650fig2.jpg" /> 
Figure 2: The layout for a 96-well source plate with seventeen Pr/Pb sets. Target number refers to a Pr/Pb set. Black arrows represent when a Pr/Pb set is added to multiple wells. NTC mixes are added to the designated wells (dark grey background). The two wells (F3, G3) with diagonal lines are unused. <a href="https://www.jove.com/files/ftp_upload/52650/52650fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52650/52650fig3.jpg" /> 
Figure 3: Schematic of specific steps in the qRT-PCR assay protocol. A-E diagram select portions of the protocol. (A) Step 1: Preparation of standard serial dilutions. (B) Step 2: Preparation of Pr/Pb and NTC working stock mixes. (C) Step 3.1: Preparation of a 96-well working stocks plate of the Pr/Pb sets and NTC mixes. (D) Step 4.1-3: Preparation of the mixes for loading the 384-well assay plate. (E) Step 4.5: Loading the 384-well assay plate. Diagrams are read from the top left to the bottom right corner. Reagents for the interferon (IFN) assay are stored at -20 oC and are listed in the left column; lines separate actions in the protocol. <a href="https://www.jove.com/files/ftp_upload/52650/52650fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52650/52650fig4.jpg" /> 
Figure 4: The human IFN expression signature in peripheral blood mononuclear cells (PBMC) differs in response to the TLR ligands used for stimulation. PBMC were stimulated with TLR ligands and RNA was harvested for qRT-PCR analysis. The geometric means of the peak responses to poly I:C (25 &#956;g/ml) at 8 hr (red squares), LPS (10 ng/ml) at 4 hr (green triangles), imiquimod (10 &#956;M) at 16 hr (purple diamonds), CpG (1 &#956;M) at 16 hr (black circles), and unstimulated control at 16 hr (blue circles) from 6 donors are shown in log10 scale as a function of expression of the HKG UBC &#916;Cq (left) or as copy number per &#956;g of RNA (right). IFN-&#945; subtypes are ordered according to the phylogenetic plot of amino acid sequence similarity. This figure was originally published in Immunology and Cell Biology27.
<img alt="Figure 5" src="/files/ftp_upload/52650/52650fig5.jpg" /> 
Figure 5: The rhesus macaque IFN expression signature in peripheral blood mononuclear cells (PBMC) differs in response to the TLR ligands used for stimulation. PBMC from three rhesus macaques (designated by the square, diamond, and triangle) were isolated from whole blood and stimulated with lipopolysaccharide (LPS) (10 &#956;g/ml), poly I:C (50 &#956;g/ml) or imiquimod (10 &#956;g/ml). Cells were harvested at 0 hr (open shapes) or after 3 hr of TLR stimulation (closed shapes) for measurement of IFN expression. Transcript levels of type I, II, and III IFN are displayed in log10 scale as a function of expression of the HKG GAPDH &#916;Cq (left) or as copy number/&#956;g RNA (right). This figure was originally published in the Journal of Interferon and Cytokine Research28.
<img alt="Table 1" fo:content-="" fo:content-width="7in" src="/files/ftp_upload/52650/52650table1.jpg" width="700" /> 
Table 1: Human IFN primer/probe set sequences and reaction information. <a href="https://www.jove.com/files/ftp_upload/52650/52650table1.jpg" target="_blank">Please click here to view a larger version of this table.</a>
<img alt="Table 2" fo:content-="" fo:content-width="7in" src="/files/ftp_upload/52650/52650table2.jpg" width="700" /> 
Table 2: Rhesus macaque IFN primer/probe set sequences and reaction information. <a href="https://www.jove.com/files/ftp_upload/52650/52650table2.jpg" target="_blank">Please click here to view a larger version of this table.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td colspan="4">Plasmid concentration (fM)</td>
		</tr>
		<tr>
			<td></td>
			<td>A</td>
			<td>B</td>
			<td>C</td>
			<td>D</td>
		</tr>
		<tr>
			<td>IFN-&#945;1</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#945;2</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#945;4</td>
			<td>2500</td>
			<td>250</td>
			<td>25</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>IFN-&#945;5</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#945;6</td>
			<td>250</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>IFN-&#945;7</td>
			<td>250</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>IFN-&#945;8</td>
			<td>250</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>IFN-&#945;10</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#945;14</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#945;16</td>
			<td>250</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>IFN-&#945;17</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#945;21</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#955;1</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#955;2</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#955;3</td>
			<td>250</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
		</tr>
		<tr>
			<td>IFN-&#946;</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>IFN-&#969;</td>
			<td>250</td>
			<td>25</td>
			<td>2.5</td>
			<td>0.25</td>
		</tr>
	</tbody>
</table>
Table 3: Standard dilution set concentration information.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>IFN Pr/Pb Sets added</td>
			<td>Water volume added (ml)</td>
		</tr>
		<tr>
			<td>NTC 1</td>
			<td>IFN-&#946;, -&#969;, -&#955;3</td>
			<td>85.7</td>
		</tr>
		<tr>
			<td>NTC 2</td>
			<td>IFN-&#945;1, -&#945;5</td>
			<td>100.0</td>
		</tr>
		<tr>
			<td>NTC 3</td>
			<td>IFN-&#945;2</td>
			<td>114.3</td>
		</tr>
		<tr>
			<td>NTC 4</td>
			<td>IFN-&#945;4</td>
			<td>114.3</td>
		</tr>
		<tr>
			<td>NTC 5</td>
			<td>IFN-&#945;7</td>
			<td>114.3</td>
		</tr>
		<tr>
			<td>NTC 6</td>
			<td>IFN-&#945;6, -&#945;8, -&#945;10</td>
			<td>85.7</td>
		</tr>
		<tr>
			<td>NTC 7</td>
			<td>IFN-&#945;14, -&#945;16</td>
			<td>100.0</td>
		</tr>
		<tr>
			<td>NTC 8</td>
			<td>IFN-&#945;17</td>
			<td>114.3</td>
		</tr>
		<tr>
			<td>NTC 9</td>
			<td>IFN-&#945;21, -&#955;1</td>
			<td>100.0</td>
		</tr>
		<tr>
			<td>NTC 10</td>
			<td>IFN-&#955;2</td>
			<td>114.3</td>
		</tr>
	</tbody>
</table>
Table 4: NTC working stock mixes information.

Watch this Scientific Journal Video about High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes at JoVE.com

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

high-throughput-quantitative-real-time-rt-pcr-assay-for-determining

Research

JoVE Journal

Immunology and Infection

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

Bead Based Multiplex Assay for Analysis of Tear Cytokine Profiles

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and protection to the underlying cells. Analysis of tears is an emerging area for the identification of biomarkers for the prediction, diagnosis, and prognosis of various ocular diseases. Tears are easily accessible and their collection is non-invasive. Therefore, advancing technologies are gaining prominence for identification of multiple analytes in tears to study changes in protein or metabolite composition and its association with pathological conditions. Tear cytokines are ideal biomarkers for studying the health of the ocular surface and also help in understanding the mechanisms of different ocular surface disorders like dry eye disease and vernal conjunctivitis. Bead based multiplex assays have the capability of detecting multiple analytes in a small amount of sample with a higher sensitivity. Here we describe a standardized protocol of tear sample collection, extraction and analysis of cytokine profiling using a bead based multiplex assay.

Analysis of tear film cytokines helps in studying various ocular diseases. Bead based multiplex assays are simple and sensitive and enable the testing of multiple targets in samples with small volumes. Here we describe a protocol for tear film cytokine profiling a using bead based multiplex assay.

Tear film is a complex mixture of lipids, proteins and minerals which covers the external surface of the eye, thereby providing lubrication, nutrition and ...

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

Label-Free Identification of Lymphocyte Subtypes Using Three-Dimensional Quantitative Phase Imaging and Machine Learning

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification of lymphocyte subtypes is important for the study of immunology as well as diagnosis and treatment of various diseases. Currently, standard methods for classifying lymphocyte types rely on labeling specific membrane proteins via antigen-antibody reactions. However, these labeling techniques carry the potential risks of altering cellular functions. The protocol described here overcomes these challenges by exploiting intrinsic optical contrasts measured by 3D quantitative phase imaging and a machine learning algorithm. Measurement of 3D refractive index (RI) tomograms of lymphocytes provides quantitative information about 3D morphology and phenotypes of individual cells. The biophysical parameters extracted from the measured 3D RI tomograms are then quantitatively analyzed with a machine learning algorithm, enabling label-free identification of lymphocyte types at a single-cell level. We measure the 3D RI tomograms of B, CD4+ T, and CD8+ T lymphocytes and identified their cell types with over 80% accuracy. In this protocol, we describe the detailed steps for lymphocyte isolation, 3D quantitative phase imaging, and machine learning for identifying lymphocyte types.

We describe a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and a machine learning algorithm. Measurements of 3D refractive index tomograms of lymphocytes present 3D morphological and biochemical information for individual cells, which is then analyzed with a machine-learning algorithm for identification of cell types.

We describe here a protocol for the label-free identification of lymphocyte subtypes using quantitative phase imaging and machine learning. Identification ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.