Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

The ddTRAP assay provides the ability to obtain a sensitive, robust, and highly quantitative measurement of telomerase enzymatic activity in cells. The ability to run up to 96 samples per run in a medium-throughput fashion offers us a great advantage. We can run the controls and replicates for proper statistical analysis on the data collected.

Immediately prior to cell lysing, thaw a frozen aliquot of NP-40 lysis buffer. Once thawed, place the lysis buffer on ice. Add PMSF protease inhibitor to the NP-40 lysis buffer to reach a final concentration of 0.2 millimolar.

Then, add 40 microliters of the lysis buffer to the tube containing a cell pellet to reach a cell equivalence of 25, 000 cells per microliter. Gently pipette the lysate up and down in order to break open the cells. Try to avoid making bubbles.

Allow the cells to lyse on ice for 30 minutes. Gently vortex the lysate every 10 minutes to prevent clusters of cell debris from forming. During the cell lysis, prepare a master mix in a microcentrifuge tube for the telomerase extension reaction, and store on ice.

After the cell lysis, pipette 48 microliters of extension master mix into each PCR tube. Add two microliters of diluted lysate to each PCR tube to reach a final cell equivalence of 50 cells per microliter, and continue the telomerase extension reaction according to the manuscript. Prepare a master mix in a microcentrifuge tube for the ddTRAP, and keep it at room temperature.

Pipette 19.8 microliters of ddPCR master mix into each PCR tube. Add 2.2 microliters of the previous extension reaction solution to each tube, leaving a total volume of 22 microliters. To set up the droplet generation cartridge, first load 20 microliters of the prepared solution into the middle sample well in the cartridge.

Gently tap the side of the cartridge to move any bubbles to the top of the solution. All wells of the cartridge must be loaded with sample prior to loading oil. Then, load 70 microliters of droplet generation oil into the left oil well.

Secure the gasket in place by tethering it to the ends of the cartridge, and place the loaded and assembled cartridge into the droplet generator. The generator shows a solid green light to inform the cartridge is placed properly. After 60 to 90 seconds, when the generator light stops blinking, the cycle is complete.

Now remove the cartridge. Gently remove the gasket from the cartridge. Use a multichannel pipette to pipette approximately 40 microliters of the newly generated droplets from the right well into a 96-well PCR plate.

Heat seal the plate with aluminum foil PCR plate seals once all the samples are loaded in the 96-well plate to prevent evaporation. Then, load the 96-well plate into a thermocycler to perform the PCR reaction. To begin, load the 96-well plate into the droplet reader.

Orient the plate properly so that the A1 sample matches that of the holder. Open the software associated with the droplet reader. Double-click the first well, A01, to open the sample well editor screen.

Click Experiment, and select the type of assay to be used from the drop-down menu. Select QX200 ddPCR EvaGreen Supermix as the correct detection method. Click Apply in the lower right-hand side of the well editor screen to save the user-defined settings to all of the highlighted wells.

Next, click on Target in the well editor screen, and click the Target drop-down menu to select either unknown, reference, or no-template control to define the type of sample. In the Sample Name section, label all the samples and click Apply. Click Run to read the plate.

Select either Columns or Rows in the Run Options screen when prompted to inform about the orientation the plate should be read in. Determine the number of accepted droplets for each sample by double-clicking on the individual wells or column and row headers to provide a table of information. For the ddTRAP, samples with 10, 000 or more accepted droplets are valid for further analysis.

Highlight the wells representing sample replicates and NTC samples. Manually set the threshold for the samples by clicking on the icon for setting thresholds on the bottom left of the screen. In this protocol, telomerase activity was measured in a cell panel consisting of nine cell lines of lung cancer and telomerase-negative fibroblasts.

A threshold was set at around 2, 000 fluorescence amplitude for all three biological replicates for SHP77, H2887, and NTC. Positive droplets had a fluorescence intensity around 6, 000 fluorescence amplitude, which formed a clear population at the top, and separate from negative droplets around 1, 100 fluorescence amplitude. The total telomerase extension products per cell equivalent between all the samples were estimated from the measured concentration of the nucleic acids, which represents the telomerase activity in lung cancer lines.

Any RNase contamination during the lysis or extension reaction steps will destroy telomerase enzymatic activity since it is a ribonucleoprotein complex. ddTRAP can be followed up with ddPCR on hTERT mRNA expression levels in the cell. The two data sets allows us to correlate expression of hTERT with telomerase activity and dig deeper into potential telomerase regulatory mechanisms such as alternative splicing.

We can explore manipulations to specific splicing factors and their effects on telomerase activity. Currently, we are designing oligonucleotide splicing modulators as potential drugs targeting telomerase activity.