Cell-Free DNA (cfDNA) Purification Kit

Synonyms

Cell-free DNA, cfDNA, circulating cell free DNA, biomarker, purification, extraction, isolation, enrichment, serum, plasma, DNA

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Overview

Cell-free DNA (cfDNA) circulates in blood plasma outside of cells, and has recently been used as a powerful sample type for disease diagnostics. Epigenetic marks found on cfDNA, such as DNA methylation, can be used as biomarkers for cancer, pregnancy, and aging, to name a few. However, this powerful method is inherently limited by the amount of cfDNA that can be isolated from a blood draw. Therefore, it is important to maximize retention of cfDNA when purifying from a sample.

The Cell-Free DNA (cfDNA) Purification Kit is designed to efficiently isolate cell-free DNA from human plasma and serum samples. Using Active Motif's fast, magnetic bead-based protocol, it is easy to isolate and concentrate cell-free DNA from fresh or frozen samples. The profile below demonstrates total elimination of high molecular weight genomic DNA from the sample, leaving behind the signature bimodal size distribution profile of cfDNA.

Cell-Free DNA (cfDNA) Purification Kit

cfDNA that has been shed into the blood retains the epigenetic signatures (DNA methylation, for example) of its past. Researchers have recently harnessed this characteristic of cfDNA to detect and monitor cancer, estimate biological age, and follow fetal DNA in pregnant mothers.

Following cfDNA extraction, global DNA methylation levels can be investigated with the Global DNA Methylation Assay - LINE-1 kit, which is a simple ELISA-based assay. The purified cfDNA can be also immunoprecipitated with MeDIP or hMeDIP kits for targeted or genome-wide analysis of methylation or hydroxymethylation.

Both the Cell-Free DNA (cfDNA) Purification Kit and a competitor magnetic bead-based cfDNA purification Kit (Competitor T) were used to isolate DNA from 600 µl of plasma from a normal human donor in triplicate using the KingFisher Flex Purification System. Results show the total DNA yield (ng) per ml plasma. The Cell-Free DNA (cfDNA) Purification Kit consistently recovered more DNA than the alternative purification method.

Automation compatibility with KingFisher^™ Flex Purification System.The Cell-Free DNA (cfDNA) Purification Kit contains enough reagents to isolate cell-free DNA from up to 100 ml or 250 ml plasma or serum. To learn more about the kit, click on the Documents, Data, or Contents tabs below.

Data

Isolate and Concentrate Cell-Free DNA

Active Motif's Cell-Free DNA (cfDNA) Purification Kit was used to isolate cell-free DNA from 5 ml of healthy human plasma. DNA was eluted in the minimum elution volume to concentrate the sample and quantified using the Qubit^™ dsDNA High Sensitivity Assay. Isolated DNA was run on a TapeStation to assess the size of the purified DNA. Results show the majority of the purified sample is cell-free DNA at the expected size of 150-200 bp.

Figure 1: Use of TapeStation to assess DNA size of isolated cell-free DNA.

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Robust and Reproducible Data

Plasma samples from four different human donors were tested in duplicate to demonstrate the reproducibility of the Cell-Free DNA (cfDNA) Purification Kit. The kit was used to isolate 1 ml plasma in each reaction. Total nanograms DNA recovered (as determined by Qubit) is shown in the table below. Results show reproducibility between replicates and the robustness of the assay to isolate between 1-100 ng total DNA per 1 ml plasma input.

Figure 2: Reproducibility of the Cell-Free DNA (cfDNA) Purification Kit.

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Kit Contents

Contents & Storage

The Cell-Free DNA (cfDNA) Purification Kit contains sufficient reagents to isolate cell-free DNA from 100 ml or 250 ml human plasma or serum. Easily process input volumes from 100 µl to 10 ml. Both fresh and frozen plasma can be used, however, fresh plasma tends to have higher yields. Plasma from blood collected with Streck Cell-Free DNA BCT^® Tubes must go through a Proteinase K treatment (not included) prior to purification to ensure optimal yields.

The Cell-Free DNA (cfDNA) Purification Kit is shipped and can be stored at room temperature. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. This kit includes the following components:

• Lysis/Binding Buffer AM2; Store at RT
• Wash Buffer AM8; Store at RT
• Elution Buffer AM6; Store at RT
• Magnetic Bead Solution; Store at RT

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