Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.
This protocol enables the detection of tumor associated mutations and currently compliments invasive surgical procedures. Another strength is the ability to use this protocol to monitor tumor mutation load overtime. The technique enables tumor genotyping without requiring actual surgical tissue biopsy which is particularly helpful for tumors in challenging locations, like the brain stem with restricted tissue access.
In the absence of repeat biopsy, longitudinal molecular data isn't available to compliment MRI. Digital PCR monitoring facilitates a molecular based supplement to diagnosis, enabling a more informed treatment decision making process. Similar methods can be used to detect the expression of genes in particular system, with conventional Fluo-4 and quencher-linked probes but with a higher sensitivity than with the quantitative PCR.
Before beginning the droplet digital PCR procedure, clean the bench space and equipment with 10%bleach and 70%ethanol and gently vortex and briefly centrifuge all of the reagents, except the droplet stabilizing oil. Confirm that the compressed nitrogen gas cylinder is attached to the droplet generating instrument and that the cylinder tank is set at 90 pounds per square inch. And 38 microliters of digital PCR reaction mixture directly into the bottom of each well of an eight well PCR tube strip, removing any bubbles with a clean pipette tip.
Add 12 microliters of pre-amplified DNA sample in triplicate to the appropriate wells of the tube strip. Gently pipette the full volume 10 times to mix the reaction mixture with the DNA sample and pipette the full volume from each well into the corresponding A through H channels of a droplet generating instrument chip. Insert a new PCR tube strip into the droplet generating instrument and scan the droplet generating instrument chip ID into the software on the instrument computer.
Then click Start the Run to begin dropletizing samples. When the dropletization is complete, remove the PCR tube strip and apply tube strip caps. Transfer the tube strip to a thermal cycler and balance the tube with another tube strip containing 80 microliters of water per well.
Then run the thermal cycler under the appropriate thermal cycling conditions. Replace the strip caps with high speed caps. When the thermal cycling is complete, transfer the tube strip to the quantification instrument, click Set Up a Run on the instrument software and place the tube strip into the quantification instrument.
Place the metal shield on top of a new quantification instrument chip, scan the chip ID into the instrument and insert the chip into the machine. Close the lid of the instrument. In the computer software, enter a name for the digital PCR run and for each of the eight channels, then select Fast Mode and click Start to begin the quantification.
To analyze the raw spectral data, launch the analyst software and open the fcs files. Under analysis view, select intact. Under sample view, click the boxes next to each of the eight samples.
The software will plot the signals for the mutant and wild type alleles along the x and y axis respectively. Use the calculated matrix function to apply for the spectral compensation on the intact droplets, as per the manufacturers instructors and adjust the axis settings under axis options. Set the x axis to a minimum of zero and a maximum of 30, 000 and the y axis to a minimum of minus 5, 000 and a maximum of 10, 000.
When droplet clusters have been identified, adjust the axis to reduce the empty space in the graph. Select the sample corresponding to the positive control tumor tissue genomic DNA to set the negative mutant and wild type gates, ensuring that the clusters are distinct and easily identified. Then right click and select Apply All Settings to Selected Samples to apply the gate settings for the positive control to all of the samples.
Under the graphical view, click multiple samples to view the plots for all of the selected samples and export the image as a tif file. Then under Workspace, select Export Analysis and save the analyzed data file as a csv file. Here, representative results for a successful detection of a mutation in a pre-amplified plasma and cerebrospinal fluid cell free DNA from two children with diffuse midline gliomas are shown.
In this experiment, a clear separation between the mutant and wild type clusters can be observed along the x and y axis respectively. Robust wild type clusters indicate that the cell free DNA extraction was successful because template DNA is present. For these patients, the mutant clusters show a mutation allelic frequency of 1.6 and 39.32 percent for the plasma and cerebrospinal fluid samples respectively in accordance with the tumor mutation status as confirmed by genomic analysis of the biopsied tumor tissue.
The negative control on the other hand shows zero mutant and zero wild type droplets indicating that there was no contamination of the PCR reaction mixture. The positive control tumor tissue genomic DNA shows the mutation being detected at the expected allelic frequency for the particular tumor sample selected. The absence of mutation detection within the plasma may not necessary mean that a patient is wild type for the mutation of interest as false negatives do occur.
For example, in this patient, although the mutation of interest was not detected, the mutation status was confirmed by genomic analysis of the tumor tissue. The PCR is a very sensitive technique that demands a frequent decontamination of the work area and equipment to avoid the acquisition of false positive data.
