The overall goal of this procedure is to quantify the levels of mRNA in individual cells using microfluidic qPCR with internal standards. This method can help answer key questions in the field of single cell biology, such as whether cell populations of cells respond to a stimulus in characteristically different ways. The main advantage of this technique is that it can measure expression of more genes than RNA FISH at a lower cost than RNA-Seq in single cells.
We first had the idea for this method when we were exploring protocols to measure gene expression in single cells by microfluidic qPCR. We wanted to modify existing methods to more accurately estimate the absolute number of different transcripts in single cells. Demonstrating the cell sorting procedure will be William Telford, the manager of the flow cytometry facility used by my lab.
After generating and purifying amplicons from genes of interest according to the text protocol, combine 18 microliters of each purified amplicon in a 2.0 milliliter low binding microcentrifuge tube, then add DNA suspension buffer up to 1, 800 microliters so that the concentration of each amplicon is five times 10 to the sixth molecules per microliter. Thoroughly vortex the 5e6 standard and divide it into 20 microliter aliquots in low binding PCR tubes. After plating MCF7 p53-Venus cells according to the text protocol, add 400 nanograms per milliliter of neocarzinostatin to the cells and treat them for three, 8.5, 14 or 24 hours.
To make lysis buffer, combine the reagents listed in this table. Transfer 92.4 microliters of the buffer into a separate tube to serve as the lysis buffer for the amplicon standards. The remaining lysis buffer will be for the cells.
Prepare and add E.coli DNA to the lysis buffer for the cells, as per the text protocol. To prepare standards, label six low binding centrifuge tubes. Add 90 microliters of DNA suspension buffer to the 5e5 tube.
Add 90 microliters of buffer containing 6.2 picograms per microliter of E.coli DNA to each of the other five tubes and keep all the tubes on ice. After removing an aliquot of 5e6 standard from storage, briefly vortex the tube, then spin it to ensure that the liquid is at the bottom of the tube, then using a low binding pipette tip, add 10 microliters of 5e6 standard to the 5e5 tube. Vortex, spin and put the tube back on ice.
Next, pipette 10 microliters from the 5e5 tube into the 5e4 tube, then briefly vortex the tube, spin it, and put the tube back on ice. Repeat the serial dilutions with each tube, receiving 10 microliters from the previous tube until all tubes have a dilution of standard. After preparing PCR tubes with lysis buffer, using a 12 channel pipette, distribute nine microliters of cell lysis buffer from the row of PCR tubes into each well of the first seven rows of a PCR plate.
Distribute seven microliters of standard lysis buffer to each well of the last row of the plate, then using low binding pipette tips, add two microliters of standard to each well of the bottom row according to the plate map. Add two microliters of 3.1 picograms per microliter of E.coli DNA into the 10 cell and 100 cell wells, then add two microliters of 6.2 picograms per microliter of E.coli DNA into the no cell wells. After programming the fluorescence activated cell sorter to sort into a 96 well plate according to the plate map, to aim the machine, seal an empty PCR plate and use the test stream to sort droplets and PBS onto the seal of the empty plate.
If the droplets do not land in the correct position, wipe them off the surface of the seal, recalibrate the cell sorter according to the machine manufacturer's instructions, and repeat until the droplets in the sort stream are deposited correctly. Harvest cells by trypsinization and re-suspend them in 0.5 milliliters of PBS with 2%FBS, then transfer to a flow cytometry tube. To sort cells, insert the tube with the cell suspension into the cell sorter, open the seal on the plate of lysis buffer, and carefully sort the cells of interest into the plate according to the plate map.
To ensure that single cells are sorted at maximum purity, run the machine in single cell mode and use standard flow cytometry gating based on forward and side scatter. Following the run, use a new sterile thermal seal to seal the plate and centrifuge it at 400 times g for four degrees Celsius for one minute, then place the plate in the thermal cycler and run the previously prepared RT-STA program. After spinning the completed RT-STA plate, use a 12 channel pipette to distribute 3.6 microliters of dilute exonuclease I into each well of the plate.
Seal and briefly spin the plate, then place the plate in the thermal cycler and run the exo I program. To measure the housekeeping gene expression in each sample, prepare 900 microliters of DNA binding dye qPCR master mix for 96 reactions using primers from one of the selected housekeeping genes. Distribute nine microliters of master mix to every well of a 96 well PCR plate, then add one microliter of sample from the sample plate to the corresponding well of the PCR plate.
Run the plate on a real time PCR machine using a thermal cycling protocol suggested by the master mix manufacturer or following a standard protocol, then analyze the samples and make a sample mixes plate map according to the text protocol. Label a plate sample mixes and then, using an eight channel pipette, distribute 3.3 microliters of master mix sample loading reagent mixture into each well of a 96 well PCR plate. For each PCR plate of samples, vortex the plate for 10 seconds and spin it at 500 times g for 10 seconds.
Remove the cover and transfer 2.7 microliters of each positive one cell sample to its corresponding well on the sample mixes plate for the one cell wells indicated on the plate map. Peel the sticker off the bottom of a new microfluidic qPCR chip, then using a syringe of control line fluid with the cap still on, push down each accumulator spring to loosen it and inject each accumulator with 100 to 200 microliters of control line fluid. Insert the chip in the loading machine and run the prime script, then using an eight channel pipette, transfer 4.5 microliters of each assay mix into the left side of the microfluidic qPCR chip.
It's critical to avoid creating air bubbles at the bottom of the wells. This will create loading problems. To prevent this, touch the pipette tips to the side of the well, then pipette only to the first stop.
Transfer 4.5 microliters of each sample mix to the right side of the microfluidic qPCR chip, then insert the chip into the loading machine and run the load mix script. When the load mix script is done, verify that there are no lines across the chip, which would indicate a loading problem, then use tape to carefully remove any dust particles from the chip, then insert the chip into the microfluidic real time PCR machine and run the data collection script. Finally, analyze the data according to the text protocol.
This figure shows the expression levels of a housekeeping gene measured by qPCR to determine which wells received a single cell. Samples that received a cell have low CT values. Samples without a cell have much higher CT values.
The wells for the amplicon standard should show evenly spaced amplification curves. There should also be clear separation in the curves for the no cell, 10 cell and 100 cell wells. Seed DNA samples representing actual cells are cherry picked from multiple sorted 96 well plates and combined in a single plate to prepare for microfluidic qPCR.
This heat map summarizes the microfluidic qPCR results. The color variation in each column shows the variability in expression of each gene between different cells. The two rows that are much darker than the rest most likely represent bad samples.
While attempting this procedure, it's important to keep your work area RNase free and DNA free, especially when making the standards and lysis buffer. The pre-PCR parts of this protocol are sensitive to contamination. After watching this video, you should have a good understanding of how to quantify expression of multiple genes of interest in individual cells.
Following this procedure, other methods, like single molecule RNA FISH can be performed to validate measurements of mRNA abundance in single cells.
