A Blood-based Test for the Detection of ROS1 and RET Fusion Transcripts from Circulating Ribonucleic Acid Using Digital Polymerase Chain Reaction

The overall goal of this procedure is the isolation and characterization by droplet digital PCR of tumor derived circulating ribonucleic acid from whole blood samples. This method can help answer key questions in the oncology diagnostics field. Such as how to detect tumor derived gene fusion transcipts from circulating RNA and blood samples.

The main advantage of this technique is that it uses highly optimized methods for the stabilization, extraction, and detection of RNA variants present at low frequency and blood samples. Blood based testing is a complement to traditional tissue biopsies with the benefit of utilizing a minimally invasive technique that can provide rapid results. Although this method can provide insight into clinically significant mutations, and NSCLC, it can also be applied to other systems where the accessible and rapid identification of circulating RNA molecules in whole blood is needed.

Circulating RNA for the reverse transcription reaction is isolated from blood samples from donors as described in the text protocol. Convert the concentrated circulated RNA sample to cDNA using a commercially available reverse transcription reaction kit, including random primers. Include a no reverse transciptase control sample and an no RNA control sample.

When the reverse transcription reaction is complete, isolate cDNA from the reaction mix, using a commercially available DNA concentrator spin column. This step facilitates the removal of enzymes, primers, and free deoxynucleotide triphosphates. If the cDNA will not be used immediately in PCR reactions, store at negative 80 degrees Celsius.

Wear a disposal lab coat and nitrile gloves while performing this procedure and a dedicated reagent preparation area. Cover the probes to protect them from light, since successive light can photo bleach the fluorescent dye attached to the probe. First prepare the PCR mixes for a final reaction volume of 20 microliters as indicated in this table, but without yet adding the cDNA.

Next, transport the mixes, covered and protected from light to a PCR clean hood, located in a separate pre-amplification area. Distribute the mixes to a PCR plate. Add the cDNA to be tested to the PCR mix.

Cover the plate with a removable plate sealer. Centrifuge the plates briefly to collect the samples at the bottom of the wells. Mix on a plate shaker, on a low setting for 10 seconds.

Centrifuge the plates briefly again to collect the samples at the bottom of the well. Remove the plate sealer prior to performing manual droplet generation. It is important to minimize bubbles in the pipette tip when loading the samples and later to transfer the droplets slowly from the cartridge to the plate to prevent droplet damage.

Carefully transfer 20 microliters of the PCR mix to the sample wells on the droplet generation cartridge. Add 70 microliters of drop generation oil. Cover with a rubber gasket and transfer the cartridge to a manual droplet generator to initiate droplet generation.

Following droplet generation, use tips recommended by the manufacturer to transfer droplets to a fresh PCR plate. Aspirate and dispense the droplets slowly, over 5 to 6 seconds each, without touching the end of the tip to the droplet cartridge or plate. Seal the plate with a foil plate sealer.

Perform PCR in the thermal cycler using the following settings. After the thermal cycler run is complete, read the plate using a droplet reader. Create a plate layout for reader software that identifies the location of controls and samples and load into the software to start the read.

The plate read results are analyzed using commercially available software. Begin by navigating to the analyze menu to view 2 dimensional or 2D Amplitude plots. Evaluate the overall quality of the data by examining the droplet data.

Using the events menu, evaluate the data for total accepted event numbers. There should be over 10, 000 events per well. If not, the data must be carefully evaluated for additional problems.

Next, check the data for abhorrent fluorescence amplitudes. Significant amplitude differences and concentration differences between replicate samples indicate poor handling or mixing of samples. Make note of droplet clusters with spray patterns on a 45 degree axis, which is indicative of poor quality droplets or problematic samples.

Examine the data for the positive control, no reverse transcriptase control and no RNA control first. Select all control samples and examine cluster quality by 2D plot for proper thresholding, a clear separation between droplet cluster should be apparent. For each assay variant, set the threshold based on control wells.

Set thresholds on 2D plots using the cross hairs tools to separate the double negative droplet population from the control gene population on the x axis, and variant gene population, if present, on the y axis. Some copies from each replicant well for a single sample. Lastly, express the test results as the number of variant copies detected.

Using cell lines, expressing SDC4-ROS1 fusion and CCDC6-RET fusion, diluted in the background of total human wild type RNA, the limit of detection as established at 0.2 percent variant frequency with each fusion variant. A preparation of 5 percent off target cell line derived RNA was not detected, demonstrating assay specificity. Analytic multiplex RNA standards were measured at high, medium, and low concentrations over three runs on the same day.

Three runs on three consecutive days, and with two independent operators. The results demonstrate accurate detection of both the fusion transcript of interest as well as an internal control gene GUSB. Each bach of clinical samples was also run with a positive control, a no reverse transcriptase control, and a no RNA template control.

Fusion variants are represented along the y axis while GUSB is on the x axis. When the batch controls were assessed over the course of 21 days, fusion positive droplets and GUSB control gene droplets were observed for ROS1 and RET in all runs. Representatives sub optimal results are shown where there is contamination within the no reverse transcriptase control, contamination within the no RNA control, sharing and coalescing of droplets, and poorly optimized PCR conditions or PCR inhibition.

Once mastered, this entire protocol can be done in approximately 12 hours. While attempting this procedure, it's important to use best practices for handling RNA and human blood specimens. Following the isolation of RNA, other methods like next generation sequencing could be performed in order to answer additional questions such as the identification of novel fusion variants.

After watching this video, you should have a good understanding of how to isolate circulating RNA from whole blood and plasma and then process the recovered RNA in order to identify rare variants in circulation such as RET and ROS1. After it's development, this technique paved the way for the detection of clinically actionable mutations from whole blood. And we have extended these methods to examine over expressed transcripts in circulation.