Introduction
Is eccDNA qPCR the Right Tool for Your Study
If your goal is to confirm specific eccDNA junctions and quantify them reliably, this service is built for you. Below are the situations where eccDNA qPCR makes sense—and what you'll get out of it.
1) Rapidly validate discovery hits
You've identified circles from whole-genome/targeted sequencing (short- or long-read) using circle-calling algorithms or circle-enriched libraries, and need a quick wet-lab check.
- You send: junction coordinates or FASTA, organism/genome build, sample type.
- You get: a junction-spanning qPCR assay and a clear yes/no with quantitative signal, plus optional amplicon sequencing to verify the break-join.
2) Screen known targets across a cohort
You're comparing conditions, lines, or tissues and want consistent numbers across samples.
- You send: target list and sample overview.
- You get: harmonized assays and a single results table you can analyze immediately (copies per ng, per µL, or per genome equivalent—your choice).
3) Track biology in models
You need to see how a defined circle changes with treatment, stress, or time.
- You send: model description and timepoints.
- You get: a sensitive, repeatable readout tied to that exact junction, with controls appropriate for your matrix.
4) Extend liquid-biopsy research
You want to follow a few circles in serum/plasma as pathway readouts.
- You send: target junctions and matrix notes (e.g., prior linear-DNA depletion).
- You get: a junction-specific assay adapted to cfDNA, with optional enrichment steps to improve signal-to-noise.
5) Prioritize targets for deeper work
You're choosing which candidates move forward to ddPCR or long-read structural analysis.
- You send: a ranked list of candidates and any constraints.
- You get: quick, comparable numbers that help you decide what's worth the next investment.
What makes a good qPCR target?
- A unique junction (not in highly repetitive sequence)
- An amplicon length suitable for qPCR
- Matrix compatibility (cells, tissues, cfDNA, or purified eccDNA)
- Not sure? Send the list—we'll flag what's assayable and suggest alternatives where needed.
Applications
What Researchers Worry About — and How We Handle It
Linear DNA noise (false positives or weak signal).
We offer enzymatic depletion options (ExoV or Plasmid-Safe DNase) to reduce linear DNA before qPCR. For matrices rich in mitochondrial DNA, we add mitochondria-aware steps (e.g., rare-cutter digestion or CRISPR-based linearization) so the assay reads the intended circle, not background. Benefit: higher signal-to-noise with minimal loss of small circles.
Junction specificity in repetitive regions.
We design outward-facing primers directly across the break-join and run in-silico genome scans to avoid paralogs/repeats. If needed, we switch to probe chemistry (e.g., short hydrolysis probes or LNA probes) to increase discrimination. Optional: Sanger of the qPCR amplicon to confirm the exact junction.
Very low-abundance targets.
Assays are tuned for short amplicons and high efficiency; we use probe chemistry where SYBR isn't clean enough, increase technical replicates when warranted, and optimize cycling to extend dynamic range—without compromising specificity.
Matrix variability (cells, tissues, cfDNA).
We choose controls that fit the matrix: minus-enrichment controls to monitor depletion steps, extraction/process controls to flag losses, and inhibition checks for plasma and other inhibitor-prone samples. Outcome: comparable results across diverse sample types.
Run-to-run comparability.
We build standard curves from plasmid/gBlock or genomic standards and include stable calibrators, so you can compare plates and batches confidently.
Bioinformatics → wet-lab handoff.
Send coordinates or FASTA from your circle-calling pipeline; we verify genome build, uniqueness, and amplicon feasibility, then return assayable targets. If a junction is not designable, we suggest nearby alternatives that preserve biological relevance.
Clear, flexible normalization.
Deliverables include raw Cq and efficiency metrics so you can choose the readout you need—copies per ng DNA, per µL extract, or per genome equivalent—without reprocessing.
Advantages
Why Quantify Specific eccDNAs
Turn discovery into decisions.
Once you've flagged candidate circles, junction-specific qPCR gives you numbers you can trust for go/no-go calls—without spinning up another discovery run.
What this readout helps you answer
- Does this junction exist in my model or matrix? Clear positive/negative with an amplicon tied to the break-join.
- How does it change with perturbation? Track fold-changes across time points, doses, or passages to see whether the circle is responsive or stable.
- Is it worth deeper investment? Use quantitative trends to prioritize which targets advance to ddPCR or long-read structural work.
- Is there a circulating signal worth following? Evaluate a small panel in serum/plasma before committing to larger cohorts.
Where this adds unique value (beyond discovery libraries)
- Targeted sensitivity on a known junction. You're measuring a single, well-defined event rather than signal averaged across a locus or a complex structure.
- Sample-sparing and fast turnaround. Minimal input and straightforward run conditions make it practical for serial sampling and screening.
- Cohort comparability. Uniform assays and controls yield a single table you can analyze across many samples, studies, and labs.
- Actionable negatives. If a junction is undetectable, you know early and can redirect resources to more promising targets.
Examples of study questions we routinely support
- Oncology models: Does the junction linked to an amplified driver persist after drug pressure?
- Stress/apoptosis models: Do small circles rise under defined stressors?
- Biofluid research: Is a selected junction detectable and stable in serum/plasma across collection protocols?
Workflow
How the Service Works
Start point options
- You already have junctions → send coordinates or FASTA, genome build, and matrix.
- You have a locus but no junction → send discovery evidence; we propose assayable break-join candidates.
Step 1 — Feasibility check (no-lab commitment from you)
- You provide: target(s) and organism/genome build.
- We do: uniqueness scan, repeat assessment, predicted amplicon, and a control plan.
- You receive: a short feasibility note with any design caveats before work begins.
Step 2 — (Optional) eccDNA enrichment plan
- When used: low-abundance circles or cfDNA matrices.
- We do: recommend enzymatic depletion for linear DNA and mitochondria-aware options if needed.
- Outcome: improved signal-to-noise without over-trimming small circles.
Step 3 — Junction-specific primer/probe design
- We do: outward (divergent) primers across the break-join, in-silico off-target screen, amplicon length tuned for qPCR; probe chemistry if the region is complex.
- You receive: sequences, expected amplicon, and design notes for approval.
Step 4 — Build & run qPCR
- We do: set up reaction chemistry (SYBR or probe), plate layout, negative and matrix-appropriate controls, and replicate strategy.
- Outcome: clean amplification focused on the intended circle junction.
Step 5 — Specificity confirmation (optional but recommended)
- We do: Sanger sequencing of the qPCR amplicon to confirm the exact break-join.
- You receive: trace files and a simple alignment snapshot.
Step 6 — Analysis & reporting
- You choose the readout: copies per ng DNA, per µL extract, or per genome equivalent.
- We deliver: raw Cq table, replicate summaries, standard-curve plots (if applicable), assay efficiency/R², control outcomes, and the final normalized results table ready for analysis.
What this workflow avoids
- Ambiguous signals from linear DNA carryover
- Non-designable targets slipping through to the bench
- Inconsistent normalization across cohorts
Before you ship samples
Please include a brief note on extraction method, any prior linear-DNA depletion, and expected target size range. This helps us align controls and chemistry from the outset.
What You Get
What You Will Receive
1) Primer/Probe Design Report
- Primer/probe sequences, expected amplicon length, GC%, Tm, and exact junction coordinates
- A simple junction schematic showing primer orientation
2) Run files & QC bundle
- Plate map and raw instrument outputs
- Amplification and (if SYBR) melt-curve summaries with NTC overview
- A concise QC note highlighting any flags
3) Quantification results
- Master results table with sample/target mapping, technical replicates, mean Cq, SD, and your chosen readout (copies per ng DNA, copies per µL extract, or per genome equivalent)
- Standard-curve summary and fit metrics when applicable
4) Specificity confirmation pack (optional)
- Sequencing trace summary and a one-page alignment snapshot confirming the break-join
5) Enrichment snapshot (if applied)
- Pre/post depletion comparison with a simple visualization of signal-to-noise improvement
6) Methods brief
- Reagents, cycling program, per-target annealing details, and the control set used
7) Data handling & ownership
- Your sequences and results remain your property; access is limited to your designated contacts
- Clear naming and metadata conventions for smooth pipeline handoff
Pick the Scope That Fits Your Study
Target Confirmation
For a quick, decisive check on one or a few junctions.
- Best for: validating sequencing-derived candidates or confirming presence in a new matrix.
- You provide: coordinates/FASTA, genome build, sample matrix.
- We deliver: junction-spanning assay(s), a concise quant result table, and optional amplicon sequencing for identity confirmation.
- Why choose this: low friction; fast go/no-go on specific targets.
Panel Screening
For comparing defined circles across cohorts, conditions, or timepoints.
- Best for: multi-sample studies, model perturbations, or longitudinal designs.
- You provide: target list, sample map, preferred normalization (copies/ng, per µL, or per genome equivalent).
- We deliver: harmonized assays, unified controls, and one clean results table aligned to your sample map.
- Why choose this: consistent numbers across many samples with minimal hands-on coordination.
Method Transfer
For groups that want to run the assay in-house after development.
- Best for: core facilities and labs standardizing a small set of circles.
- You provide: target priorities and internal platform details.
- We deliver: design files, cycling programs, control set, and a concise SOP so your team can reproduce the assay as-is.
- Why choose this: keeps the assay close to the bench while preserving design rigor.
Add-ons (apply to any package)
- Junction identity check: Sanger of the qPCR amplicon.
- Linear-DNA depletion: enzyme-based enrichment plan for low-abundance targets or cfDNA matrices.
- Data handoff format: XLSX/CSV plus optional JSON to plug into your LIMS or pipeline.
- Reference materials: optional oligo mix or control template for future in-house runs.
Compare Methods
qPCR vs. ddPCR vs. Sequencing — Pick the Right Fit
Use qPCR when you already know the junction(s) you want to track.
- Strengths: fastest setup for predefined targets; adaptable to cells, tissues, and cfDNA; easy to scale across many samples; flexible normalization (copies/ng, per µL, or per genome equivalent).
- Limits: relative or calibrated quantitation; detection of extremely rare molecules can be constrained by background and total input.
- Typical use: validate candidates, screen cohorts, monitor dynamics in models.
Use ddPCR when you need absolute quantitation at very low copy numbers.
- Strengths: partitioning reduces background, giving high precision for scarce targets or small fold-changes; results are in copies per reaction without standard curves.
- Limits: higher assay complexity per target and generally higher per-sample cost; panel size is tighter than qPCR.
- Typical use: confirm low-abundance circles, resolve subtle changes, or set absolute baselines.
Use eccDNA sequencing when you need discovery or structural context.
- Strengths: finds new circles, resolves break-joins and larger architectures, and reveals heterogeneity beyond a single junction.
- Limits: less efficient for routine tracking of known targets; data analysis and depth requirements are higher.
- Typical use: initial discovery, structural validation, and hypothesis generation for which junction(s) to follow with qPCR or ddPCR.
Decision guide (quick):
- Known junctions, many samples → qPCR
- Known junctions, very low copy or tiny effect sizes → ddPCR
- Unknown junctions or structural questions → sequencing
If you're unsure, share your target list and sample plan—we'll recommend a practical path (including mixes: sequencing for discovery → qPCR for screening → ddPCR for critical low-copy confirmations).
Samples
Sample Guidance
- Matrices (RUO): cell pellets, primary tissues (fresh-frozen/stabilized), serum/plasma cfDNA, or purified eccDNA. No clinical submissions.
- Prep tips: cells—wash/snap-freeze; tissues—prefer fresh-frozen/stabilized; serum/plasma—EDTA tubes, minimize hemolysis; avoid repeated freeze–thaw.
- Extraction: standard column/bead kits are fine. Tell us if you used linear-DNA depletion or expect high mtDNA.
- Include with shipment: sample map, species & genome build, brief method notes, expected circle size (if known), preferred readout (copies/ng, per µL, or per genome-equivalent).
- Safety & privacy: de-identified research samples only; declare any chemical/biological hazards.
FAQ
Practical Questions Researchers Ask
- What if I don't have exact junction sequences yet?
- How many targets or samples can you accommodate?
- What if my input is limited (cfDNA, tiny biopsies)?
- Can you support non-human species?
- How do you avoid mitochondrial DNA interference?
- Can I run the assay in-house later?
- What will the data look like when delivered?
Get Started
Ready To Move Forward?
1) Request a quote
Use the short form so we can scope the assay precisely.
- Organism & genome build (e.g., GRCh38, mm10)
- Sample matrix (cells, tissues, serum/plasma cfDNA, purified eccDNA)
- Targets (# of junctions; names/IDs if applicable)
- Have junction sequences? Yes/No (upload BED/FASTA/coordinates if yes)
- Preferred readout (copies/ng DNA, copies/µL extract, or per genome equivalent)
- Optional add-ons (junction Sanger confirmation, linear-DNA depletion)
2) Secure file upload
Send BED/FASTA/coordinates and any discovery evidence (reads/contigs/notes). Include a simple target-to-sample map if you have one. We keep files private and scoped to your project.
3) Talk to a scientist (optional)
A brief scoping call to align on target designability, controls, and plate layout. Bring any constraints (sample amounts, matrix quirks, probe vs. SYBR preference).
4) Ship samples (only after digital review)
Once targets and controls are agreed, ship de-identified research samples with your sample map and method notes. We'll confirm receipt and proceed per the agreed scope.
References
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Kong, X., Wan, Sj., Chen, Tb. et al. Increased serum extrachromosomal circular DNA SORBS1circle level is associated with insulin resistance in patients with newly diagnosed type 2 diabetes mellitus. Cell Mol Biol Lett 29, 12 (2024).
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Cen, Y., Fang, Y., Ren, Y. et al. Global characterization of extrachromosomal circular DNAs in advanced high grade serous ovarian cancer. Cell Death Dis 13, 342 (2022).
