This protocol can be used to accurately measure the number of copies of mitochondrial DNA present in individual cells and the level of certain types of mitochondrial DNA mutation. Previous methods can accurately make these measurements in tissue samples containing many cells. The main advantage of this technique is that we can make the same measurements in single cells.
This method is of particularly use to those studying mitochondria and will have wide applications in this field. Effective isolation and lysis of single cells is key to this technique. Previous experience with single cell sorting would be very helpful.
To begin, prepare the master mix for the PCR without the sample DNA on ice, as described in the text. After vortexing briefly, distribute the required volume of the prepared master mix to each well of a droplet generation PCR 96-well plate, arranging the samples in full columns. Next, add the master mix to any empty well in incomplete columns for using them as non-template controls.
Then add the input DNA to each well to make the total volume of each well 22 microliters. Seal the plate with an adhesive plate seal and place it on a shaker at 2000 RPM for one minute. Then centrifuge the plate at 1000 times G for one minute at four degrees Celsius before placing it on ice.
For droplet generation, first ensure that the droplet generator is powered on and check whether a bottle of droplet generating oil is loaded to position E of the instrument deck. Then click Configure Sample Plate on the touch screen. After selecting all columns on the sample plate containing the samples or blanks, click on Okay to confirm the plate configuration.
Next, load the droplet generator cartridges to position B on the instrument deck so that all lights turn green and place an empty tip waste trough into position C.Then load filter tip boxes with lids removed to position D on the instrument stage so that all lights turn green. Next, remove the adhesive seal before loading the sample plate to position F of the instrument deck so that the light turns green. Also load an empty droplet collection 96-well plate placed into a cool block, pre-chilled at minus 20 degrees Celsius, at position G so that the light turns green.
Click Start Droplet Generation on the instrument touch screen and the droplet generator lid will close automatically. Once droplet generation is complete, check the sample collection plate visually to confirm that a droplet emulsion layer is present above the oil phase in each well. Next, power on the plate sealer and set the temperature to 180 degrees Celsius and seal time to five seconds.
Allow the machine to reach the operating temperature. Then place the sample collection plate on the plate holder of the plate sealer and place a fresh foil seal on the top of the plate with the red line facing upwards. When the plate sealer has reached its operating temperature, press eject on the instrument touch screen.
After placing the plate holder in the drawer of the plate sealer, press seal to close the drawer. Once the sealing is complete, the drawer will automatically open. Remove the sealed plate and place it on the cold block.
Then turn off the plate sealer. For running the PCR, place the sealed droplet collection plate in a PCR machine with a 96 deep-well block. After the PCR is complete, power on the droplet reader and the connected computer.
Load the plate with the post-PCR samples into the droplet reader plate holder. While keeping the foil lid on the sample plate, place the cover on the top of the holder and secure it in place with black locking clips. Next, to open the droplet reader lid, press the open/close button.
Then load the droplet reader plate holder with the sample plate into the reader chamber and place it securely on the magnetic base. Press the open/close button again to close the lid. Open the analysis software interface.
Select the Add Plate tab, click Add Plate, and then Configure Plate. In the Plate Information tab, enter a plate name, select Supermix for Probes, No DUTP from the Supermix Dropdown menu, and enter a file name under Save Data File As.In the Well Selection tab, highlight the wells to be analyzed and click Include Selected Wells. Then in the Well Information tab, select the wells to be annotated.
Select Direct Quantification from the Experiment Type dropdown menu. Then populate the sample description, sample type, and target name boxes, and add well notes and or plate notes as required. Then click Apply.
Once the plate configuration is complete, click Start Run. For result analysis, select the Data Analysis tab and open the file to be analyzed from the My Data Files menu. Then click the 1D Amplitude tab for single-color experiments, or the 2D Amplitude tab for two-color experiments.
Next, select the wells that require thresholding. Use the Threshold Line Mode tool to manually apply the threshold in the clear space between the positive and negative populations on the amplitude plot, ensuring that the crosshair is placed such that the four droplet populations are clearly separated. Once the threshold is applied for all samples, export the results as a csv file for further analysis by clicking on the Data Table tab, Import/Export, and selecting Export Visible Data to CSV.
Enter a suitable file name and click Save. The PCR and droplet reading procedures used in this protocol demonstrated the presence of clearly-separated populations of positive and negative droplets, both in the fam and hex channels. Mitochondrial DNA populations positive for both the probes could also be distinguished from single cell lysates.
This method also showed that for samples with deletions in the mitochondrial DNA, a mixed population of double-positive droplets and droplets positive for only the non-deleted portion of the DNA are present. This method has been successfully used to count the mitochondrial DNA copy number per cell in a variety of mammalian cells, including mouse oocytes and primary mouse embryonic cells. Furthermore, in cells with high deletion heteroplasmy, the population positive for a single probe was found to be significantly bigger than the double-positive population.
However, in cells with low-deletion heteroplasmy, both populations were of similar size. So accurate results always ensure that the droplet generation has been successful and that thresholding has been appropriately applied using the 1D or 2D amplitude view. Unused lysate can be used for other single cell assays, such as pyrosequencing, which can measure single nucleotide polymorphism heteroplasmy.
The development of this technique has allowed us to make accurate measurements of mitochondrial DNA copy number, and deletion heteroplasmy in individual cells, which was previously not possible.
