July 15, 2022 | PCR Solutions | Digital PCR

Top 10 FAQs on sensitive mutation analysis of cfDNA by dPCR

Your cfDNA mutation analysis questions answered

Digital PCR offers advantages in cancer research, enabling the accurate quantification of targets of interest in liquid biopsy (cfDNA) samples. It can routinely detect rare mutations in a high background of wild-type cfDNA. This webinar, presented by our scientific experts Simon Hertlein and Oezlem Karalay, and product expert Daniel Heinz Löfgren, introduced "The power of two: automated sample preparation on the EZ2 Connect and analysis using the QIAcuity Digital PCR System." In this webinar, they described an automated workflow that begins with high-volume sample processing and ends with ultra-low mutation detection to produce reliable results.

If you couldn’t attend but would like to view the recording, you can sign up to watch it here.

Here are the answers to some of the most common questions we received about complex liquid biopsy sample analysis.

Do samples have to be collected without EDTA tubes?
We have tested the most commonly used blood collection tubes for plasma generation, namely EDTA, ACD-A, PAXgene Blood ccfDNA and Streck Cell-Free DNA and observed no compatibility issues. However, we do recommend using a stabilizing blood collection tube such as the PAXgene Blood ccfDNA Tube especially if the blood cannot be processed promptly. The non-crosslinking stabilization solution in PAXgene Blood ccfDNA Tube helps prevent the release of intracellular DNA into the plasma and maintains constant ccfDNA levels during sample transport and storage before processing.

What's your recommendation about the storage conditions of the unprocessed samples for cfDNA studies? 
If using non-stabilizing blood collection tubes such as EDTA or ACD-A, we recommend processing the samples within a few hours and storage at 2-8°C in the meantime. Stabilizing blood collection tubes such as the PAXgene Blood ccfDNA Tube allow storage at room temperature for up to 10 days. Generally, we recommend plasma/serum generation as quickly as possible, with mid-to-long-term storage at -20°C. It is also advisable to avoid repeated freeze-thaw cycles.

What about cfRNA for fusion detection? Do you have experience with cfRNA? There is a stabilization tube for cfRNA, is this really necessary?
We currently do not have a kit for the EZ2 available for cfRNA extraction. For manual extraction of cfRNA, the QIAamp ccfDNA/RNA or exoRNeasy kits can be used. In blood, most cfRNA outside extracellular vesicles (EVs) are present as very short fragments and appear to be derived mainly from blood cells. In contrast, RNA derived from inside the EVs is much better preserved and has a much more diverse origin. 

Stabilization of blood samples for extraction of cfRNA and EV-RNA should mainly prevent the release of RNA and EVs from blood cells after blood draw, and in our experience, the PAXgene Blood ccfDNA Tube (RUO) can come handy.

Do you need different kits for different amounts of plasma volumes on the EZ2 Connect?
No, you can use the same kit for the three existing protocol versions for 2, 4 or 8 ml of plasma. Every single-use cartridge contains enough reagents for up to 8 ml sample input.

What is the minimum elution volume for the EZ2?
We currently only have one elution volume of 70-75 µl. However, we are working on a reduced elution volume targeting below 50 µl.

For preparing cfDNA from plasma, what volume of plasma is recommended for use in downstream applications such as dPCR and NGS?
This depends on the scientific question you are seeking answers for. If (rare) mutations are supposed to be detected, the more plasma you use, the higher the sensitivity in your downstream application of choice. Depending on sample availability, we recommend harnessing the maximum sample processing capacity of the EZ1&2 ccfDNA Kit of 8 ml plasma.

We often face problems associated with poor sample quality in cfDNA-based studies. What can you say about QIAcuity's sensitivity concerning this issue?
Poor sample quality is often associated with the presence of contaminants or PCR inhibitors in the samples, which can reduce amplification efficiency and result in inaccurate quantification of targets. Absolute quantification of targets in dPCR is based on end point PCR signal measurements and Poisson statistics. DNA templates are distributed over thousands of partitions, and each partition receives zero, one or more target molecules. PCR amplification of target molecules then takes place independently in each partition. At the end of PCR, signals from each partition indicate the presence or absence of target molecules within the partitions. Subsequently, an absolute amount of target molecules in the original sample is estimated by Poisson statistics, which takes into account the number of partitions that produce PCR signals as well as the empty partitions without PCR signals. Therefore, independent of amplification efficiency, sub-optimal or delayed amplification of target sequences within the partitions is detected as a signal and included in the statistical calculation of absolute copies present in a sample. This allows for highly sensitive and accurate quantification of targets in poor-quality samples by dPCR, even in the presence of inhibitors. 

QIAGEN sample preparation solutions like the EZ1&2 ccfDNA Kit discussed in this webinar are optimal for achieving amplifiable DNA for sensitive downstream assays.

What design strategies do you suggest for achieving specificities <0.1% (e.g., 0.001%)?
dPCR LNA Mutation Assays are composed of LNA-containing primers and probes, which increase the sensitivity and discrimination power between wild-type and mutant sequences. In addition, the incorporation of LNA oligos provides higher Tm values in shorter probe and primer lengths compared to DNA oligos. This allows optimal assay design targeting shorter amplicon regions for highly fragmented templates such as cfDNA. 

What are your recommended steps for optimizing assays? For example, using additives like betaine, DMSO, etc.
We do not recommend introducing any changes to the QIAcuity 4x Probe Master Mix. All proprietary chemical components in the QIAcuity 4x Probe Master Mix are essential for delivering the highest performance in the absolute quantification of target regions in the genome. Assay portfolios for various applications on QIAcuity are validated using the QIAcuity platform and therefore need no further optimization under recommended settings. However, homebrew assays may require optimization. A few changes to the reaction conditions can be introduced, i.e., the concentration of the assay mix, annealing temperature, and cycling conditions can be further optimized.

What is the maximum volume of sample that can be loaded into a single dPCR reaction? What percentage of this volume is actually analyzed? Does the volume of the analyzed sample affect the reaction's sensitivity?
The first factor that impacts sensitivity is template addition volume, because the more sample added to a reaction, the more template will be present for analysis. Depending on the dPCR system, the template volume added to a reaction can range from 6 to 26 μl. Secondly, the percentage of the sample analyzed affects sensitivity. This varies across different systems and is referred to as ‘dead volume’ or ‘non-analyzed volume’. A 'dead volume' is the fraction of a PCR reaction that is loaded onto a dPCR plate or chip but is not transferred into the reaction compartments and is therefore not analyzed. The QIAcuity Nanoplate 26K allows for the analysis of 21.6 μl, resulting in a 46% non-analyzed volume. We recently generated an application note to demonstrate that template addition volume and template analyzed volume are more important factors than dead volume in determining the relative sensitivity of currently available dPCR systems. Please read the application note for more information on how higher template addition volumes can overcome any limitations that dead volume may have on dPCR sensitivity.

Power up your cancer research workflow using this complementary approach to flexible automation on the EZ2 Connect and the QIAcuity Digital PCR System. 

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