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

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.

The overall goal of this experiment is to observe multiple gene expression in single cells. This method can help answer key questions in the immunology field, such as heterogenity of molecular signatures within a cell population, or a rare molecular signature. The main advantage of this technique is that specific molecular signatures with at least 48 different genes can be assessed in many cells at the same time.

Begin this procedure by preparing a pre-amplification mix for 48 reactions in a 1.5 milliliter tube. Add to the tube 240 microliters of specific retrotranscription buffer, 62.4 microliters of low EDTA TE buffer, and 9.6 microliters of Taq DNA polymerase. Using a electronic pipette, distribute 6.5 microliters of the pre-amplification mix to each of 48 wells in a 96-well, single-cell plate.

Next, prepare a 0.2x assay mix in a 1.5 milliliter tube. Add to the tube 1.4 microliters of each primer. Adjust the final volume to 140 microliters with low EDTA TE buffer.

Using an electronic pipette, distribute 2.5 microliters of the 0.2x assay mix to the 48 wells in the 96-well, single-cell sorting plate, containing the pre-amplification mix. Seal the plate with a cover film. Vortex the plate, and spin it at 280 times g for one minute.

Single hepatic innate lymphoid cells, or ILCs, will be sorted by fluorescence-activated cell sorting. Begin this procedure by using an empty 96-well plate as a test. Position the test plate on the plate holder with the a one well on the left and toward the experimenter.

Adjust the plate holder to obtain a drop in the center of the a one well with verification beads. When properly adjusted, place the 96-well, single-cell sorting plate containing the assay mix and the pre-amplification on the plate holder. Draw the plate layout, and sort one cell per well of the gated population.

The proper positioning of each cell on the 96-well, single-cell sorting plate is essential, and a plate layout should be kept on a spreadsheet software. Leave one well containing 0.2x assay mix and pre-amplification mix without cells, as a no-input control. Optionally, leave two rows of six wells for cDNA dilution as controls for primer efficiency.

Immediately after the single cell sorting, vortex, and spin down the 96-well, single-cell sorting plate. Place the plate in the thermocycler. Perform reverse transcription and pre-amplification as indicated.

Pre-amplification of specific target genes on sorted single cells is required in order to have enough material. Dilute the pre-amplified samples by adding 36 microliters of low EDTA TE buffer into each well. To begin this procedure, prepare 191 microliters of a Master Mix by pipetting 175 microliters of a qPCR Master Mix, and 17.5 microliters of the sample loading reagent into a 1.5 milliliter tube.

On a new 96-well plate, distribute 3.6 microliters of the Master Mix to each of 48 wells. This is the 96-well sample plate. Transfer 2.9 microliters of pre-amplified cDNA from the 96-well, single-cell sorting plate to the new 96-well sample plate, keeping the same position for each sample between the two plates.

Prepare a 96-well assay plate by distributing three microliters of assay loading reagent to each of 48 wells on a new 96-well plate. Add three microliters of primers to each well. The proper positioning of each primer in the 96-well assay plate is essential.

Keep a plate layout on a spreadsheet software. To begin this procedure, place the integrated microfluidic circuit, or IFC, on the bench, and check the valves using a syringe. Remove the cap of the syringe, place it perpendicular to one valve, and press firmly.

The O-ring should move. Fill the chip with control line fluid. After repeating the previous steps with the second valve, remove the black film from the bottom of the chip.

Load the chip into the IFC controller. On the IFC controller screen, select prime, and then run. Next, eject the chip, and reseal the black film on the bottom of the chip.

Using an eight channel pipette, transfer five microliters from the 96-well assay plate to the a one, or left side, of the chip. Change tips for each well of the chip. Avoid creating bubbles, and if bubbles appear, use 10 microliter tips to remove them.

Fill the left side of the chip as indicated. In the same way, fill the right side of the chip using five microliters from the 96-well sample plate. For the success of this experiment, it is essential that the IFC chip is properly filled for proper loading into the IFC controller.

Remove the blue film from the bottom of the chip, and load the chip into the IFC controller. On the IFC controller screen, select load mix, and then run. When completed, eject the chip, and reseal the blue film on the bottom of the chip.

To run the chip, first select the data collection software on the microfluidic qPCR computer. Once it starts, select new run. Select eject and load the chip.

After setting up the software as described in the text protocol, select start run. The reaction will take approximately 90 minutes. To begin data analysis, open the realtime PCR analysis software, select file, and open, find the experiment folder, and select chip run dot b m one file.

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. Name the samples.

Go to sample setup and select new SBS 96. Click on mapping, and select the sample layout according to the 96-well sample plate layout. Copy and paste the sample layout design from the spreadsheet software.

Define the pasted layout as sample name. Use the same procedure to name the assays. Enter the primer names in detector setup, and define the pasted layout as detector name.

Click on analysis view and analyze. Select file, and export, and save as heat map results. Using flow cytometry, hepatic ILC populations were sorted based on widely expressed ILC markers, and three distinct populations were defined.

A properly loaded single cell, multiplex gene expression chip should appear with straight lines and rows, with each reaction chamber filled and in the same dimension. An improperly loaded chip will have empty lines and rows of reaction chambers, as well as bending lines. This fluorescein amidite figure of a properly loaded chip shows differences in reaction chamber brightness appearing after a few cycles.

Reaction chambers with an amplification signal appear brighter than reaction chambers with no or low amplification signals. After proper cell sorting, pre-amplification, and loading, the ILC population appeared heterogeneous for gene expression in the liver of adult, wild type mice. On the left is a heat map without modifications.

On the right is the modified heat map obtained after sample and assay name definition. Using online software, cell-specific gene expression signatures and cell population relationships were identified. Each line represents a gene, and each row represents the same cell, and the three cell populations are represented in blue, red, and green.

One mastered, this technique can be done in nine hours, if it is performed properly. While attempting this procedure, it is important to carefully keep track of the plate's orientation for cell and primer distributions. After its development, this technique paved the way for researcher to explore rare and specific molecular signatures in cell populations.

After watching this video, you should have a good understanding on how to assess multiple gene expression in many single cells at the same time, after proper cell sorting, pre-amplification, and loading.