The overall goal of this method is to detect rare transcript variants in fresh-frozen tissues by chip-based digital PCR. This method can help answer key questions in the cancer research field, such as the detection of rare transcript variants that can be involved in carcinogenesis. The main advantage of this technique is that it allows the detection of low-abundant targets, improving precision and reproducibility with respect to the gold standard quantitative PCR.
Though this method, can provide insight into the detection of a low number of cDNA molecules in human fresh-frozen tissues, it can also be applied to other kinds of biological samples. Generally, individuals new to this method will struggle because it is necessary to acquire some skill in preparing the chips and defining parameters for the analysis. To begin, let the master mix and the assay thaw at room temperature for at least 20 minutes.
Then, load 0.5 milliliter tubes with six microliter aliquots of the cDNA samples. Also, make a no template control tube. Next, gently vortex the master mix and aliquot 8.7 microliters per reaction into a sterile tube.
Then, for each reaction, add 0.87 microliters of the custom assay primer, and add 1.83 microliters of nuclease-free water. Mix the combination gently, and transfer 11.4 microliters to each tube containing cDNA, and to the no template control. Then, mix the tubes gently, and pool the tube contents using pulse centrifugation.
For optimal results, load the chips as soon as possible. Plug in the chip loader, and wait until the indicator light turns green. Then, prepare the immersion fluid syringe by gently pulling the plunger to one to two millimeters, then remove the cap and add the tip.
Next, take note of the code written on the lid of a new chip. Then, hold the lid carefully by the side, peel away the protective film, and position it in the lid nest with the sticky side up. Now, press down the lever to open the clamp, and carefully load the chip into the chip nest.
Next, put a new loading blade into the loader. Push it gently to ensure that it is firmly in place. Now, transfer 14.5 microliters of reaction mix onto the loading blade.
Do not make air bubbles and do not deflect the blade. Then, press the black loading button to distribute the volume onto the chip. It is important to confirm the loading blade is in place.
Then, while filling the blade, do not depress the pipette to the second stop, to avoid introducing bubbles. Also, do not deflect the blade with the tip. Proceed by using the syringe to transfer about 20 drops of immersion fluid onto the chip surface.
Take care not to touch the surface with the tip, and completely cover the surface without overflowing it. Next, rotate the loader arm to make the lid come into contact with the chip and press down for 15 seconds. Then, press the lid button to release the chip, and return the loader arm to its resting position.
Now, open the clamp and hold the assembled chip at a 45 degree angle to carefully dispense the immersion fluid through the fill port via a syringe. Then, rotate the chip slightly to remove air bubbles, and then remove any excess fluid with a sterile wipe. Avoid spilling immersion fluid on the border of the chip.
If that happens, carefully remove excess before sealing. Finally, seal the chip case by gently peeling away the label on the lid, and then blocking the fill port for at least five seconds. Now, use the chip within two hours, and until then, store it in the dark.
To set up the reaction, open the lid and install the adapters on both blocks, even when a single block is used. Then, place a chip on the sample blocks and orient its fill ports towards the front of the thermal cycler, and elevate the port slightly. Use empty chips to balance the two blocks.
Next, lay the thermal pad over the setup to completely cover the chips. Then, program the cycles, close the lid, and start the reaction. When the reaction is over, turn off the thermal cycler, remove the thermal pads, and then remove the chips.
Let the chips thaw to room temperature for at least 10 minutes in the dark, and analyze them within an hour. Clean the chip surface with isopropanol while inspecting them for leaks or other issues. To save the data, insert a USB memory stick into the detector system, where the data can be saved.
Then, open the chip tray of the detector system and load the chip face up. Close the tray, and wait 30 seconds for the data to be processed. Then, remove the chip, and repeat the process for the next chip.
Next, move the USB stick from the detector to a computer and transfer the files. Open the cloud-based software, then create a project and import all of the data files for the chips of interest. Then, under Define Chips tab, select the dye and assay used.
Determine if the chip is acceptable by visualizing it in the Review Data tab. If the sample was loaded well, at least 13, 000 points should be usable. Next, look at the scatter plot for the selected chip.
There, apply a threshold defined by the assay type. For fluorescein amidite reporter dye signal use a threshold of 6, 000. Now, remove any dubious signals that could result in false positives.
Select the questionable spot on the scatterplot using the lasso tool, and select the Undetermined option. All the remaining positive spots indicate the presence of cDNA copies of the rare target analyzed. Using the procedure presented here, we checked for the expression of the rare transcript variant CDH1a in gastric fresh-frozen tissues.
If the chip had multiple bubbles, a single large bubble, or fewer than 13, 000 data points, it was omitted from the analysis. No positive signals above the selected threshold of 6, 000 for the FAM channel were present in the no template control. The yellow points indicate no amplification.
A fine chip analysis filtered the low-quality data points and eliminated the risk of ambiguous results. When a blue signal was located, indicating amplification, if it was at the very borders of the chip or around bubbles, the signal was omitted. Similarly, to further prevent the risk of false positives, signals above the selected FAM threshold and on the far right of the plot were likely to be aspecific and were also filtered out.
21 paired normal and cancer tissue samples and 11 additional tumor samples were analyzed. Overall, the rare transcript CDH1a was detectable in 15 out of 32 tumors. By contrast, none of the 21 control tissue samples showed the presence of this rare transcript.
After watching this video, you should have a good understanding of how to detect a rare transcript variant in fresh-frozen tissue by chip-based digital PCR. Once mastered, this technique can be done in six to eight hours, depending on the number of samples, if it is performed properly. While attempting this procedure, it's important to remember to accurately load the sample into the blade.
After its development, this technique paved the way for researchers in the field of molecular diagnostics to explore rare sequence variants in cancer patients.
