Name: Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations (Video) | JoVE Journal
Uploaded: 2017-01-19T13:00:00.000+00:00
Duration: 10 min 23 s
Description: Learn single-cell multiplex RT-qPCR to analyze gene expression heterogeneity in rare lymphoid populations. This sensitive method enables simultaneous assessment of 48 genes across many cells for immunology research.

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5 minutes

This work was supported by the Institut Pasteur, INSERM, Universit&#233; Paris Diderot and by the Minist&#232;re de la Recherche (to S.C.); the Association pour la Recherche sur le Cancer (to S.C. and R.G.); the REVIVE Future Investment Program and the Agence Nationale de Recherche (ANR; grant ''Twothyme'' to A.C.); ANR grant ''Myeloten'' (to R.G.); and the Institut National du Cancer (Role of the immune microenvironment during liver carcinogenesis, to R.G.). We acknowledge the Center for Human Immunology and Cytometry platform at Institut Pasteur for their support.

All animal experiments were approved of by the Pasteur Institute Safety Committee in accordance with the French Agriculture Ministry and the EU guidelines.
1. Prepare a 96-well Single-cell Sorting Plate
<ol>
	<li>Prepare pre-amplification mix in a 1.5 ml tube by adding 5.0 µl of specific retro-transcription buffer, 1.3 µl of low ethylenediaminetetraacetic acid (EDTA) TE buffer (10 mM TE solution, pH 8, and 0.1 mM EDTA solution; 0.2 µM filtered), and 0.2 µl of Taq DNA polymerase per well (Figure 1a).</li>
	<li>On a 96-well plate, distribute 6.5 µl of pre-amplification mix in each well (up to 48 wells). This plate is the 96-well single-cell sorting plate. 
	NOTE: Using an electronic pipette is recommended for this protocol. It allows for precise and reproducible volume measurements and saves time.</li>
</ol>
<img alt="PCR setup diagrams with pre-amplification mix, assay mix, and dilution analysis in 96-well plates." src="/files/ftp_upload/54858/54858fig1.jpg" data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1: 96-well plate loading procedure. The loading of the 96-well plates should be done very carefully, as it will impact the rest of the experiment. (a) On the 96-well single-cell sorting plate, the pre-amplification mix is distributed first, followed by the 0.2x assay mix. (b) The record of each single-cell position should be kept on a spreadsheet. A well without a cell is called a "no input" well and can be used as a control. Two rows can be spared to control primer efficiency with cDNA dilution (sequential one-in-ten dilutions from the equivalent of 105 cells to one cell). (c) On the 96-well assay plate, the assay loading reagent is distributed first, followed by the addition of the primers. Do not forget to keep a layout of each primer position. <a href="http://cloudfront.jove.com/files/ftp_upload/54858/54858fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Prepare a 0.2x assay mix in a 1.5 ml tube by adding 1.4 µl of each primer (primers 20x; up to 48 different probes; see Table 1). Adjust the final volume to 140 µl with low EDTA TE buffer.</li>
	<li>Distribute 2.5 µl of the 0.2x assay mix to the 48 wells in the 96-well single-cell sorting plate containing the pre-amplification mix. 
	NOTE: The final volume in each well of the 96-well sample plate should be 9 µl.</li>
	<li>Seal the plate with a cover film. Vortex the plate and spin it down at 280 x g for 45 sec. Store the 96-well single-cell sorting plate at -20 °C or use it immediately.</li>
</ol>
2. Single Cell Dissociation
<ol>
	<li>Using a CO2 delivery system, euthanize a mouse by hypoxia. Fix the mouse on a dissection plate and open the peritoneal cavity longitudinally using scissors.</li>
	<li>Recover ILCs from the liver.
	<ol>
		<li>Prior to liver isolation, flush the liver-circulating cells.
		<ol>
			<li>To bring out the portal vein, move the small and the large intestine to the left side of the mouse; the portal vein appears as the largest vein in the peritoneal cavity.</li>
			<li>With a 10 ml syringe and a 0.45 mm needle, flush the liver with phosphate-buffered saline medium (PBS) through the portal vein. To facilitate liver flushing, cut the gastric artery with scissors.</li>
			<li>To remove the gallbladder, pinch the base of the gallbladder with forceps and separate it from the liver with scissors.</li>
			<li>To isolate the liver, pinch the base of the liver with forceps, and using scissors, separate the liver from the rest of the peritoneal cavity.</li>
			<li>Transfer the liver in a potter tube with 5 ml of Roswell Park Memorial Institute medium (RPMI) 2% fetal calf serum (FCS).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Dissociate the liver with a mechanic dissociation using a pestle. Transfer the medium to a 15 ml tube. Bring the total volume to 14 ml with RPMI 2% FCS. Leave the cell suspension on ice for 15-20 min to decant the hepatocytes.</li>
	<li>Recover the supernatant (without the hepatocytes) in a new 15 ml tube using a 1 ml pipette (12 ml of supernatant can be expected). Spin down at 120 x g for 7 min at 10 °C. Discard the supernatant after centrifugation.</li>
	<li>Resuspend the pellet obtained in step 2.4 in 14 ml of density gradient medium (e.g., Percoll 40%). Spin down at 600 x g for 20 min at 20 °C. Remove the supernatant by aspiration.</li>
	<li>Resuspend the pellet in 1 ml of potassium acetate (ACK). Leave at room temperature for 1 min. After 1 min, bring the total volume to 14 ml with Hank's Balanced Salted Solution (HBSS) 2% FCS. Spin down at 120 x g for 7 min at 10 °C. Discard the supernatant.</li>
	<li>Stain the cells.
	<ol>
		<li>Resuspend the pellet in 300 µl of biotinylated antibody mix (see the Materials Table; add all biotinylated antibodies mentioned) and transfer the cells in to 1.5 ml tube. Leave it in the dark for 20 min at 4 °C.</li>
		<li>Bring the total volume to 1 ml with HBSS 2% FCS. Spin down at 120 x g for 7 min at 10 °C. Resuspend the pellet in 300 µl of fluorochrome-coupled antibody mix and fluorochrome-conjugated streptavidin (see the Materials Table; add all fluorochrome-coupled antibodies mentioned). Leave it in the dark for 20 min at 4 °C.</li>
	</ol>
	</li>
	<li>Bring the total volume to 1 ml with HBSS 2% FCS. Spin down at 120 x g for 7 min at 10 °C. Remove the supernatant using a 1 ml pipette. Resuspend the pellet in 1 ml HBSS and propidium iodide (Pi; 1:4,000). 
	NOTE: When using the widely expressed ILC cell-surface markers, 3 populations are defined: NKp46+ IL-7Rα-, NKp46+ IL-7Rα+, and NKp46- IL-7Rα+. All populations are sorted lineage- CD3- CD4- CD45.2+.</li>
</ol>
3. Single-cell Fluorescence-activated Cell Sorting (FACS)
<ol>
	<li>Use FACS to sort single cells13. 
	NOTE: Different cell types can be sorted on the same 96-well single-cell sorting plate (Figure 2).
	<ol>
		<li>Use FACS to sort one cell per well containing the 0.2x assay mix and the pre-amplification mix on the 96-well single-cell sorting plate. Use a freshly prepared 96-well single-cell sorting plate or thaw it if stored at -20 °C.
		<ol>
			<li>Put a sealed 96-well plate on the FACS plate holder. Use an empty 96-well plate as a test. Position the plate with the A1-well on the left and toward the experimenter.</li>
			<li>Adjust the plate holder to obtain a drop in the center of the A1-well with verification beads.</li>
			<li>Repeat step 3.1.1.2 with all the wells in line A.</li>
			<li>Remove the seal from the 96-well plate and sort 100 verification beads per well. Check for drop formation in the center of the wells.</li>
			<li>When properly adjusted, place the 96-well single-cell sorting plate on the plate holder. Draw the plate layout and sort 1 cell per well. 
			NOTE: The proper positioning of each cell on the 96-well single-cell sorting plate is essential. A plate layout should be kept on a spreadsheet software. The plate layout will be used in the next several steps (Figure 1b)14. Leave one well containing 0.2x assay mix and pre-amplification mix on the 96-well sample plate without cells. This well is used as a no-input control. Leave two rows of 6 wells for a cDNA dilution. These wells are used as controls for primer efficiency (Figure 1b).</li>
			<li>Store the 96-well single-cell sorting plate at -20 °C or use it immediately.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Pre-amplification
<ol>
	<li>Use a fresh or thawed 96-well single-cell sorting plate obtained in step 3.1.1.6. Vortex the plate and spin it down (280 x g for 45 sec).</li>
	<li>Place the 96-well single-cell sorting plate on the thermocycler. Perform reverse transcription and pre-amplification as per the mentioned program in Figure 3 and Table 2.</li>
</ol>
<img alt="PCR thermal cycling diagram, 40-95°C, preamplification, showing temperature steps and cycle durations." src="/files/ftp_upload/54858/54858fig3.jpg" data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3: Pre-amplification program. In order to have enough material, pre-amplification of specific target genes on sorted single cells is required. The 96-well single-cell sorting plate is loaded on a thermocycler to follow the pre-amplification program. The pre-amplification products are then diluted with low EDTA TE buffer and can be used immediately or frozen at -20 °C. <a href="http://cloudfront.jove.com/files/ftp_upload/54858/54858fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Dilute the pre-amplified samples by adding 36 µl of low EDTA TE buffer into each well.</li>
	<li>Seal the plate with a cover film. Vortex the plate and spin it down (280 g for 45 sec). Store the pre-amplified, 96-well single-cell sorting plate at -20 °C or use it immediately.</li>
</ol>
5. Prepare a 96-well Sample Plate
<ol>
	<li>Prepare 191 µl of master mix by adding 175 µl of qPCR master mix and 17.5 µl of sample loading reagent.</li>
	<li>On a new 96-well plate, distribute 3.6 µl of master mix in each well (up to 48 wells). This is the 96-well sample plate.</li>
	<li>Transfer 2.9 µl of pre-amplified cDNA from the 96-well sample plate to the new 96-well plate. Keep the same position for each sample between the 96-single cell sorting plate and the 96-well sample plate.</li>
	<li>Seal the plate with a cover film. Vortex the plate and spin it down (280 x g for 45 sec). Place the new 96-well plate on ice protected from light. Store the 96-well sample plate at -20 °C or use it immediately.</li>
</ol>
6. Prepare a 96-well Assay Plate
<ol>
	<li>On a new 96-well plate, distribute 3 µl of assay loading reagent in each well (up to 48 wells). This plate is called the 96-well assay plate.</li>
	<li>Add 3 µl of primers to each well. Keep a layout on a spreadsheet software (Figure 1c and Table 1; use all primers listed in Table 1). The proper positioning of each primer on the 96-well assay plate is essential for the next several steps.
	<ol>
		<li>If the number of samples is below 48, add water instead of primers to unused wells.</li>
	</ol>
	</li>
	<li>Seal the plate with a cover film. Vortex the plate and spin it down (280 x g for 45 sec). Store the 96-well assay plate at -20 °C or use it immediately. 
	NOTE: To avoid repeated freezing and thawing of the primers, distribute the primers for the pre-amplification mix and for the 96-well assay plate at the same time.</li>
</ol>
7. Single-cell Gene-expression Chip
<ol>
	<li>Place the integrated microfluidic circuit (IFC) on the bench and check the valves using the capped syringe. Open the syringe, place it perpendicular to the valve, and press firmly. The O-ring should move. Fill the chip with control line fluid (0.3 ml of tuberculin).</li>
	<li>Repeat step 7.1 with the second valve. 
	NOTE: No liquid should be spilled over the chip.</li>
	<li>Remove the blue film from the bottom of the chip. Load the chip into the IFC controller. On the IFC controller screen, select "PRIME" and then "RUN." The control of the microfluidic lines last 10 min.</li>
	<li>Eject the chip and re-seal the blue film on the bottom of the chip.</li>
</ol>
<img alt="Microfluidic circuit diagram; assay and sample plates, integrated chip, process flow visualization." src="/files/ftp_upload/54858/54858fig4.jpg" data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4: Single-cell multiplex gene-expression chip loading. These steps require great precision, especially during the transfer of the 96-well plate to the single-cell multiplex gene-expression chip. To avoid loading errors and misplacements, it is highly recommended to work sequentially. The volume taken for each transfer should be controlled during the pipetting process. Finally, it is important to avoid any bubbles and to remove them in case of formation. <a href="http://cloudfront.jove.com/files/ftp_upload/54858/54858figfourlarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Using an 8-channel pipette, transfer 5 µl from the 96-well assay plate on the A1 side (left side) of the chip. Fill the left side of the chip, as indicated in Figure 4. Be careful to always draw the same volume into the tips.
	<ol>
		<li>Do not create bubbles. If bubbles appear, remove them using 10 µl tips. Change tips for each well of the chip.</li>
	</ol>
	</li>
	<li>Repeat step 7.5 on the right side of the chip using 5 µl from the 96-well sample plate.</li>
	<li>Remove the blue film from the bottom of the chip. Load the chip into the IFC controller. On the IFC controller screen, select "RUN SCRIPT" and then "RUN." The loading of the microfluidic lines lasts 45 min.</li>
	<li>Eject the chip and re-seal the blue film on the bottom of the chip.</li>
</ol>
8. Run the Chip
<ol>
	<li>On the microfluidic qPCR computer, select the "Data Collection" software. Once it starts, select "New Run."</li>
	<li>Select "Eject," remove the blue film from the bottom of the chip, and load the chip. Place the chip to get the A1 well on the left and toward the experimenter.</li>
	<li>On "Project setting," select "None" and then "Next."</li>
	<li>Select "New chip run," "New chip directory" (create a folder for the experiment), and "Next."</li>
	<li>On "Gene expression" select, "Passive reference = ROX" (carboxy-x-rhodamine) and "Single probe;" select the correct filter according to the primers. Select "Next."</li>
	<li>Select "Browse" and select the correct program according to the primers used. 
	NOTE: "Default-10min-Hotstart.pcl" is the most commonly used program for single-cell multiplex RT-qPCR.</li>
	<li>Select "Start Run". The reaction takes approximately 90 min.</li>
	<li>Once finished, select "Eject-Done."</li>
</ol>
9. Data Analysis
<ol>
	<li>Open the "Real-Time PCR Analysis" software, select "File" and "Open," find the experiment folder, and select "ChipRun.bml file."</li>
	<li>Click on "Analysis View," "Results Table," and "Heat Map View;" boxes marked with an "X" are below the threshold detection level and/or had bad amplification curves.</li>
	<li>Name the samples. Go to "Sample Setup" and select "New SBS 96." Click on "Mapping" and select "…" Copy and paste the sample layout design from the spreadsheet software. Define the pasted layout as "Sample Name."</li>
	<li>Name the assays. Repeat step 9.3 with the primer names in "Detector Setup." Define the pasted layout as "Detector Name."</li>
	<li>Click on "Analysis view" and "Analyze;" the sample and primer names will be attributed automatically to the samples and primers (Figure 7).</li>
	<li>Calculate Ct, Delta Ct, and Fold Change for each sample. Use a housekeeping gene as an internal control (here, Gapdh): 
	ΔCt = Ctsample - Ctinternal control 
	Fold Change = 2-(ΔCtsample-ΔCtnegative control)
	<ol>
		<li>Click on "Detector Setup" and select the well with no input control (use the housekeeping gene as an internal control to normalize gene expression). Select "Editor," "define reference," and "Update."</li>
		<li>Select all wells to which the above-defined internal control will be applied. Select "Editor" and "define as Test." Select an appropriate reference gene and click "Update."</li>
		<li>Select "Sample Setup" to select the negative control well. Select "Editor" and define "Reference." Click "Update."</li>
		<li>Select "Analysis View" and "Analyze;" the Delta Ct and Fold Change will automatically be calculated.</li>
	</ol>
	</li>
	<li>Export the data. Select "File" and "Export, save as "Table Results," select the destination folder, and hit "Save" and "Exit."</li>
	<li>Repeat step 9.7, but instead of "Table Results," save as "HeatMap Results."</li>
</ol>

<ol><li>Sun, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Bead-Based+Microfluidic+Approach+to+Integrated+Single-Cell+Gene+Expression+Analysis+by+Quantitative+RT-PCR%5BTitle%5D)+AND+%22RSC+Adv%22%5BJournal%5D)">A Bead-Based Microfluidic Approach to Integrated Single-Cell Gene Expression Analysis by Quantitative RT-PCR.</a> RSC Adv. 5, 4886-4893 (2015).</li>
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<li>Spits, H., Artis, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Innate+lymphoid+cells+-+a+proposal+for+uniform+nomenclature%5BTitle%5D)+AND+%22Nat.Rev.+Immunol%22%5BJournal%5D)">Innate lymphoid cells - a proposal for uniform nomenclature.</a> Nat.Rev. Immunol. 13 (2), 145-149 (2013).</li>
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The authors have nothing to disclose.

This protocol describes how to obtain exhaustive gene expression information at the clonal level. Here, we investigated liver ILC compartment heterogeneity. After single-cell sorting of different ILC populations (based on widely expressed ILC surface markers), samples were pre-amplified for specific pre-selected genes. Then, the obtained cDNA and primers were loaded onto a multiplex RT-qPCR microfluidic chip. Finally, we obtained the expression of 48 different genes from 48 single cells. Gene expression results were anal...

Over the past few years, innate lymphoid cells (ILCs) have been increasingly investigated. Despite their lack of antigen-specific receptors, they belong to the lymphoid lineage and represent important sentinels for tissue homeostasis and inflammation. ILCs are currently divided into three groups based on their expression of specific transcription factor combinations and on their ability to produce cytokines6,7.
ILCs contribute to numerous homeostatic and pathophysiological situations in diverse organs via specific cytokine production8,9. To be able to understand the role of these cells, it is important to determine the various ILC subpopulations per organ and to identify their developmental relationships. In addition, phenomena of plasticity between the different subsets have been related. By studying the heterogeneity of the cells present in one organ, it is possible to delimit their stage of maturation and to distinguish their specific functions.
To illustrate the technique of single-cell multiplex RT-qPCR, hepatic ILCs were chosen, with a particular emphasis on their heterogeneity within the same ILC group (type 1 ILC)10. First, through the use of flow cytometry, three distinct ILC populations were characterized in the liver. Group 1 ILC represents around 80% of the innate effectors, while the two other populations are rare hepatic ILC populations (less than 5% of the innate effectors). Those populations were sorted using widely expressed cell-surface markers of ILC populations. As a result, sorted ILC populations in the liver look broadly similar one to another.
Single-cell multiplex RT-qPCR has emerged as one of the best techniques to promptly investigate the heterogeneity of these populations11. Two main characteristics are determined by taking advantage of the single-cell multiplex RT-qPCR technique. First, by looking at the clonal level, it is possible to recover cell-specific gene expression for comparison between cells that apparently display similar developmental stages. Then, by looking at a pre-selected combination of gene expression, we will determine new gene signatures based on simultaneous gene expression patterns at one time point. These aspects permit the collection of a wide variety of expression data for a large number of cells, even on rare populations, since the technique is performed at the clonal level. Thereby, ILC heterogeneity in the liver can be adequately assessed.
Next, by sorting all cells with a global ILC phenotype, a wide overview of the multiple-gene expression of the liver ILC populations is obtained, even though they represent extremely rare populations. A microfluidic-based chip allows experimentation with even a small amount of cell material. As a consequence, the gene expression profiles of rare cell populations can be obtained. Using online gene signature analysis software, cell population clusters and potential cell relationships can be investigated. Consequently, functional tests can be performed to validate the clustering data at the in vivo level.
Tens of gene expressions could be assessed concomitantly on hundreds or more single cells on the same chip3,11,12. Design of the assay is the longest and most important part of the experiment. The determination of the genes relevant to the hypothesis to be tested is paramount to obtain relevant results. Secondly, internal control (such as known surface markers used for sorting) and specific controls are needed. This is crucial to test the primer amplification specificity, the efficiency of the amplification, and the absence of primer competition. Therefore, working with single-cell multiplex RT-qPCR is a timesaving technique, as multiple-gene expression of a cell is assessed at the same time.
Using the same chip and mix of reagents for all cells limits the possible errors of manipulation and allows for reproducibility between samples. Altogether, the different aspects of single-cell multiplex RT-qPCR allows for the production of highly reliable results at the clonal level, with a great level of sensitivity for a wide variety of samples and genes. The obtained results offer powerful and robust data for biostatistical tests.
This can be achieved due to the microfluidic aspect of the method, which allows for work on very small amounts of material and leads to exhaustive results. Finally, using online software, it is possible to compare the desired populations.

<table><tbody><tr><td>Cells Direct One Step qRT-PCR kit</td><td>Applied Biosystems&amp;nbsp;</td><td>11753100</td><td>Primer probe detection kit.Contains 2x reaction mix, SSIII Platinium enzyme.</td></tr><tr><td>Low TE EDTA Buffer</td><td>Affymetrix</td><td>75793 100ML</td><td></td></tr><tr><td>96-well plates</td><td>Thermofisher Scientific</td><td>AB 1100</td><td>96-well plates adapted for cell sorting and thermocycling</td></tr><tr><td>Cover film</td><td>Dominique Dutscher</td><td>106570</td><td>aluminium cover film; avoid contamination and evaporation</td></tr><tr><td>Actb</td><td>Thermofisher Scientific</td><td>Mm00607939_s1</td><td>20x primer</td></tr><tr><td>Aes</td><td>Thermofisher Scientific</td><td>Mm01148854_s1</td><td>20x primer</td></tr><tr><td>Ahr</td><td>Thermofisher Scientific</td><td>Mm00478932_s1</td><td>20x primer</td></tr><tr><td>Bcl2</td><td>Thermofisher Scientific</td><td>Mm00477631_s1</td><td>20x primer</td></tr><tr><td>c-myc</td><td>Thermofisher Scientific</td><td>Mm00487804_s1</td><td>20x primer</td></tr><tr><td>Cbfb</td><td>Thermofisher Scientific</td><td>Mm01251026_s1</td><td>20x primer</td></tr><tr><td>Cd27</td><td>Thermofisher Scientific</td><td>Mm01185212_s1</td><td>20x primer</td></tr><tr><td>Cd49a</td><td>Thermofisher Scientific</td><td>Mm01306375_s1</td><td>20x primer</td></tr><tr><td>CD49b</td><td>Thermofisher Scientific</td><td>Mm00434371_s1</td><td>20x primer</td></tr><tr><td>Cxcr5</td><td>Thermofisher Scientific</td><td>Mm00432086_s1</td><td>20x primer</td></tr><tr><td>Cxcr6</td><td>Thermofisher Scientific</td><td>Mm02620517_s1</td><td>20x primer</td></tr><tr><td>Eomes</td><td>Thermofisher Scientific</td><td>Mm01351985_s1</td><td>20x primer</td></tr><tr><td>Ets1</td><td>Thermofisher Scientific</td><td>Mm01175819_s1</td><td>20x primer</td></tr><tr><td>Foxo1</td><td>Thermofisher Scientific</td><td>Mm00490672_s1</td><td>20x primer</td></tr><tr><td>Gapdh</td><td>Thermofisher Scientific</td><td>Mm03302249_s1</td><td>20x primer</td></tr><tr><td>Gata3</td><td>Thermofisher Scientific</td><td>Mm00484683_s1</td><td>20x primer</td></tr><tr><td>Gm-csf</td><td>Thermofisher Scientific</td><td>Mm01136644_s1</td><td>20x primer</td></tr><tr><td>Hes1</td><td>Thermofisher Scientific</td><td>Mm01342805_s1</td><td>20x primer</td></tr><tr><td>Hprt</td><td>Thermofisher Scientific</td><td>Mm00446968_s1</td><td>20x primer</td></tr><tr><td>Id2</td><td>Thermofisher Scientific</td><td>Mm01293217_s1</td><td>20x primer</td></tr><tr><td>Il-12rb2</td><td>Thermofisher Scientific</td><td>Mm00711781_s1</td><td>20x primer</td></tr><tr><td>Il-18r1</td><td>Thermofisher Scientific</td><td>Mm00515178_s1</td><td>20x primer</td></tr><tr><td>Il-1rl1</td><td>Thermofisher Scientific</td><td>Mm00434237_s1</td><td>20x primer</td></tr><tr><td>Il-22</td><td>Thermofisher Scientific</td><td>Mm001226722_s1</td><td>20x primer</td></tr><tr><td>Il-23r</td><td>Thermofisher Scientific</td><td>Mm00519943_s1</td><td>20x primer</td></tr><tr><td>Il-2ra</td><td>Thermofisher Scientific</td><td>Mm01340213_s1</td><td>20x primer</td></tr><tr><td>Il-2rb</td><td>Thermofisher Scientific</td><td>Mm01195267_s1</td><td>20x primer</td></tr><tr><td>IL-7r</td><td>Thermofisher Scientific</td><td>Mm00434295_s1</td><td>20x primer</td></tr><tr><td>Klr5</td><td>Thermofisher Scientific</td><td>Mm04207528_s1</td><td>20x primer</td></tr><tr><td>Lef1</td><td>Thermofisher Scientific</td><td>Mm00550265_s1</td><td>20x primer</td></tr><tr><td>Ncr1</td><td>Thermofisher Scientific</td><td>Mm01337324_s1</td><td>20x primer</td></tr><tr><td>Nfil3</td><td>Thermofisher Scientific</td><td>Mm01339838_s1</td><td>20x primer</td></tr><tr><td>Notch1</td><td>Thermofisher Scientific</td><td>Mm00435249_s1</td><td>20x primer</td></tr><tr><td>Notch2</td><td>Thermofisher Scientific</td><td>Mm00803069_s1</td><td>20x primer</td></tr><tr><td>Rora</td><td>Thermofisher Scientific</td><td>Mm01173766_s1</td><td>20x primer</td></tr><tr><td>Rorc</td><td>Thermofisher Scientific</td><td>Mm01261022_s1</td><td>20x primer</td></tr><tr><td>Runx3</td><td>Thermofisher Scientific</td><td>Mm00490666_s1</td><td>20x primer</td></tr><tr><td>Tbx21</td><td>Thermofisher Scientific</td><td>Mm01299453_s1</td><td>20x primer</td></tr><tr><td>Tcf3</td><td>Thermofisher Scientific</td><td>Mm01175588_s1</td><td>20x primer</td></tr><tr><td>Tcf7</td><td>Thermofisher Scientific</td><td>Mm00493445_s1</td><td>20x primer</td></tr><tr><td>Tle1</td><td>Thermofisher Scientific</td><td>Mm00495643_s1</td><td>20x primer</td></tr><tr><td>Tle3</td><td>Thermofisher Scientific</td><td>Mm00437097_s1</td><td>20x primer</td></tr><tr><td>Tsc22d3</td><td>Thermofisher Scientific</td><td>Mm01306210_s1</td><td>20x primer</td></tr><tr><td>Tnfrsf11a</td><td>Thermofisher Scientific</td><td>Mm00437132_s1</td><td>20x primer</td></tr><tr><td>Tox</td><td>Thermofisher Scientific</td><td>Mm00455231_s1</td><td>20x primer</td></tr><tr><td>Zbtb16</td><td>Thermofisher Scientific</td><td>Mm01176868_s1</td><td>20x primer</td></tr><tr><td>Zbtb7b</td><td>Thermofisher Scientific</td><td>Mm00784709_s1</td><td>20x primer</td></tr><tr><td>qPCR Master mix&amp;nbsp;</td><td>Applied BioSystems</td><td>P/N 4304437</td><td></td></tr><tr><td>2x Assay Loading Reagent</td><td>Fluidigm</td><td>P/N 85000736</td><td>Specific density medium to load assays in multiplex RT-qPCR microfluidic chip.&amp;nbsp;</td></tr><tr><td>2x Sample Loading Reagent</td><td>Fluidigm</td><td>P/N 85000735</td><td>Specific density medium to load samples in multiplex RT-qPCR microfluidic chip.&amp;nbsp;</td></tr><tr><td>48.48 mutliplex RT qPCR microfluidic chip</td><td>Fluidigm</td><td>BMK-M-48.48</td><td>48.48 Dynamic Array IFC for Gene Expression;chip for single cell multiplex RT-qPCR reaction</td></tr><tr><td>48.48 mutliplex RT qPCR microfluidic chip controller</td><td>Fluidigm</td><td>89000020</td><td>48.48 IFC Controller; control the chip internal fluidic system, load samples and assays in reaction chambers</td></tr><tr><td>mutliplex RT qPCR microfluidic thermocycler</td><td>Fluidigm</td><td>GE48.48</td><td>48.48 Dynamic Array IFC thermocycler</td></tr><tr><td>96-well plates</td><td>Thermofisher Scientific</td><td>AB 1100</td><td>96-well plates adapted for cell sorting and thermocycling</td></tr><tr><td>C57Bl/6 mice</td><td>Janvier</td><td>C57Bl/6 miceJ@RJ</td><td></td></tr><tr><td>10 ml syringe</td><td>BD Biosciences</td><td>309639</td><td></td></tr><tr><td>PBS</td><td>Life Technologies</td><td>14040174</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies</td><td>24020133</td><td></td></tr><tr><td>RPMI</td><td>Life Technologies</td><td>61870044</td><td></td></tr><tr><td>FCS</td><td>CVFSVF000U</td><td>Eurobio Abcys</td><td>Standard fetal calf serum</td></tr><tr><td>Potter tube</td><td>N/A</td><td></td><td></td></tr><tr><td>15 ml tube</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>1.5 ml tube</td><td>Sigma-Aldrich</td><td>T9661-1000EA</td><td></td></tr><tr><td>FACS machine</td><td>N/A</td><td></td><td></td></tr><tr><td>centrifuge&amp;nbsp;</td><td>Thermofisher Scientific</td><td>75004538</td><td></td></tr><tr><td>1,000 &amp;micro;l tips</td><td>Fisher Scientific</td><td>10313272</td><td></td></tr><tr><td>P1000&amp;nbsp;</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>Percoll</td><td>Dominique Dutscher</td><td>17-0891-01</td><td></td></tr><tr><td>Facs tube</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>anti-CD8 Biotin mouse antibody</td><td>Sony</td><td>1103520</td><td>lineage antibody</td></tr><tr><td>anti-CD19 Biotin mouse antibody</td><td>Sony</td><td>1177520</td><td>lineage antibody</td></tr><tr><td>anti-TCRab Biotin mouse antibody</td><td>BioLegend</td><td>109204</td><td>lineage antibody</td></tr><tr><td>anti-TCRgd Biotin mouse antibody</td><td>BD Biosciences</td><td>553176</td><td>lineage antibody</td></tr><tr><td>anti-Ter119 Biotin mouse antibody</td><td>BD Biosciences</td><td>553672</td><td>lineage antibody</td></tr><tr><td>anti-Gr1 Biotin mouse antibody</td><td>BD Biosciences</td><td>553125</td><td>lineage antibody</td></tr><tr><td>anti-CD45.2 PerCPCy5.5 mouse antibody</td><td>BioLegend</td><td>109828</td><td></td></tr><tr><td>anti-IL7ra PeCy7 mouse antibody</td><td>ebioSciences</td><td>25-1271-82</td><td></td></tr><tr><td>anti-CD3 BV510 mouse antibody</td><td>BD Biosciences</td><td>563024</td><td></td></tr><tr><td>anti-CD4 BV786 mouse antibody</td><td>BD Biosciences</td><td>563727</td><td></td></tr><tr><td>anti-NKp46 PE mouse antibody</td><td>ebioSciences</td><td>12-3351-82</td><td></td></tr><tr><td>Streptavidin</td><td>Sony</td><td>2626025</td><td></td></tr><tr><td>Propidium Iodide</td><td>Sigma-Aldrich</td><td>P4864-10ML</td><td></td></tr><tr><td>Electronic pipette</td><td>Eppendorf</td><td>4986000017</td><td></td></tr><tr><td>Combitips 0.1 ml</td><td>Eppendorf</td><td>30089405</td><td></td></tr><tr><td>Multichannel pipette</td><td>Rainin</td><td>L8-10XLS+</td><td></td></tr><tr><td>Accudrop</td><td>BD Biosciences</td><td>345249</td><td>verification beads for FACS</td></tr></tbody></table>

single-cell gene expression using multiplex rt-qpcr to characterize heterogeneity of rare lymphoid populations

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

Lymphoid populations display great diversity in gene expression. In this protocol, liver ILC compartment heterogeneity was investigated using single-cell multiplex RT-qPCR gene expression. Unlike other gene expression techniques, single-cell multiplex RT-qPCR gene expression allows work on several populations, even the rarest, at the same time. This specificity, coupled with a high sensitivity at the clonal level, allows for the investigation of differences in gene signatures within a pop...

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

single-cell-gene-expression-using-multiplex-rt-qpcr-to-characterize

Research

JoVE Journal

Genetics

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

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

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

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings.

Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Here we describe a robust method of determining immune cell identity and purity through epigenetic signatures detected using quantitative PCR (qPCR). DNA demethylation at a specific locus serves as a unique identifier for a particular cell type and allows for identification of CD8+, regulatory, or Th17 T cells.

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Learn single-cell multiplex RT-qPCR to analyze gene expression heterogeneity in rare lymphoid populations. This sensitive method enables simultaneous assessment of 48 genes across many cells for immunology research.

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

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