Handling cfDNA
cfDNA quality control
Consider the following parameters when assessing the quality of cfDNA purified from cell-free biofluids:
- The efficiency of the cfDNA purification and the yield
- The size pattern of the extracted cfDNA
- The absence of any inhibitors of downstream enzymatic processes such as PCR or NGS library preparation
- The absence of nucleases
- The presence or absence of gDNA from cellular contamination or hemolysis
cfDNA qualification by electrophoresis. Blood was collected in EDTA and PAXgene Blood ccfDNA Tubes. Paired tubes were processed directly (t0) or after storage for 7 days (t7d) at 25°C. cfDNA extraction was performed automated on the QIAsymphony instrument (QS) using tube-specific kits and protocols. (A) Plasma from stored EDTA tubes showed an increase in apoptotic gDNA fragments. Overlay of cfDNA electropherogram profiles from one exemplary donor. Quantification of cfDNA is evaluated by the assay using a pre-set region from 50–700 bp. Indicated are the lower marker, mononucleosomal peak and high molecular weight (HMW) DNA. (B) Detail of indicated region from panel A. Basic QC for standard samples
The amount of cfDNA that can be purified and the amount of residual inhibitors that remain after purification can vary from sample to sample. When analyzing cfDNA from standard human serum or plasma samples, we recommend performing quality control assessments on all samples – to monitor the purification yield and absence of PCR inhibitors – and to identify any potential problems in the sample set before qPCR or NGS experiments. For studies involving a very large number of samples, at least a subset should be quality controlled.
Tip: You can confirm successful and high-quality urine collection and stabilization by analyzing the size of the extracted cfDNA. cfDNA will be visible on a profile at a fragment size of about 200 bp.
cfDNA quantification
Quantitative, real-time PCR using a multi-copy target gene with a known and stable copy number per genome (e.g., GAPDH or ẞ-actin) is the most accurate method for determining the concentration of small amounts of cfDNA. Alternatively, you can use 18S rDNA as a target (1). We don’t recommend spectrophotometric quantification (e.g., Nanodrop) because the amount of cfDNA in plasma and urine is usually too low for reliable measurements. Fluorometric (e.g., Qubit) or electrophoretic quantification can also be unreliable for cfDNA due to its short nucleic acid fragments. Use designated cfDNA assays for electrophoretic quantification.
Preferred cfDNA quantification methods
✔ Real-time qPCR: short amplicon (<100 bp) of housekeeping gene✔ Digital PCR (e.g., using QIAcuity)
Comparison of methods for cfDNA quantification. cfDNA was isolated from six donors using various sample collection tubes and cfDNA extraction methods (manual or automated on QIAsymphony). cfDNA quantification using qPCR methods (Investigator® Quantiplex Pro Kit or an 18S rDNA qPCR assay) are reliably homogenous in contrast to cfDNA quantification by Qubit, which results in highly variable quantification results.