cfDNA Purification Kit (Magnetic Beads)

The cfDNA Purification Kit (Magnetic Beads) was developed for cell free DNA (cfDNA) enrichment by separating genomic DNA contamination from isolated cfDNA samples.

Many diagnostic technologies for detection of disease signals in cfDNA begin with isolation and purification of DNA from liquid biopsy that include urine, plasma, cerebrospinal fluid. The most widely explored biotechnology is assays used to detect cancer-derived plasma cfDNA. Silica-based magnetic bead cfDNA isolation kits can reliably extract total DNA from plasma, but typically yield a large variation in cfDNA that includes the presence of genomic DNA that often depends on tumor stage, tumor size, or healthy status individuals. Most of the commercial cfDNA isolation kits can’t specifically recover the cfDNA while leaving the high molecular weight genomic DNA behind. The presence of genomic DNA can lead to decreased sensitivity or inconsistent results in downstream applications such as next-generation sequencing (NGS), PCR, QPCR, and digital PCR etc.

Therefore, an additional purification step to enrich cfDNA before downstream methods helps to improve signal from fragments that originate from cancer cells. A proportion of cancer-derived cfDNA fragment signals are below 100 bp and are often not detectable except by qPCR or single-stranded DNA based library preparation for NGS (1, 2, 3). Furthermore only 1% of cancer-derived fragments are found above 400 bp (1, 2). Capture of size-selected fragments between 90-150 bp improved detection of cancer by 2-4 fold (4). Furthermore, TF-bound and protected cfDNA fragments are also being investigated for active cancer-specific signals down to 35-80 bp (5, 6).

This kit uses Dual Solid Phase Reversible Immobilization (SPRI) technology for cfDNA purification. Most Dual SPRI procedures do NOT recover fragments below 100 bp. The kit can be used for the enrichment of cfDNA isolated from liquid biopsies, plasma, serum, and urine. The kit separates cfDNA (50-500 bp) and genomic DNA, and recovers of 90% of the cfDNA without the high molecular weight genomic DNA with high efficiency. Fragments at 500 bp and above may also be retained. Both the 50-500 bp and >500 bp DNA fractions can be used for downstream applications such as single-stranded or double stranded NGS library prep, qPCR, ddPCR, and other methods.

Features

• Separation of cfDNA and genomic DNA; Recovery of both types of DNA
• Recovery of cfDNA (50-500 bp)
  • As short as 50 bp can be recovered
• Recovery of high molecular weight genomic DNA
• Removal of unwanted components and other impurities
• Automation friendly

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Examples of cfDNA purification. Both cfDNA and genomic DNA can be recovered separately.

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The range of recovered small DNA fragments is from 50 to 500 bp. The input DNA are mixtures of sheared small DNA fragments and intact genomic DNA. The ratios of sheared DNA fragments versus genomic DNA are indicated.

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Recovery rates of cfDNA and genomic DNA.

Many diagnostic technologies for detection of disease signals in cfDNA begin with isolation and purification of DNA from liquid biopsy that include urine, plasma, cerebrospinal fluid. The most widely explored biotechnology is assays used to detect cancer-derived plasma cfDNA. Silica-based magnetic bead cfDNA isolation kits can reliably extract total DNA from plasma, but typically yield a large variation in cfDNA that includes the presence of genomic DNA that often depends on tumor stage, tumor size, or healthy status individuals. Most of the commercial cfDNA isolation kits can’t specifically recover the cfDNA while leaving the high molecular weight genomic DNA behind. The presence of genomic DNA can lead to decreased sensitivity or inconsistent results in downstream applications such as next-generation sequencing (NGS), PCR, QPCR, and digital PCR etc.

Overview

• Purification and enrichment of intact urinary exosomes for functional studies
• Isolate all sizes of RNA, including microRNA, irrespective of size or GC content, without bias.
• Versatile urine input volumes
• No phenol extractions, Proteinase K treatment, nor carrier RNA required
• No time-consuming ultracentrifugation, filtration nor special syringes are required
• No precipitation reagents nor overnight incubation required
• Compatible with urine from most species
• Pure exosomes are purified and are free from any other RNA-binding proteins
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin
• The purified RNA is suitable for a variety of downstream applications, including Small RNA Sequencing. Find out more information on Norgen’s NGS services

Norgen’s Exosome Purification and RNA Isolation Kits constitute all-in-one systems for the purification of exosomes and the subsequent isolation of exosomal RNA from different urine sample volumes. The purification is based on spin column chromatography that employs Norgen’s proprietary resin. These kits are designed to isolate all sizes of RNA, including microRNA, irrespective of size or GC content, without bias. These kits provide a clear advantage over other available kits in that they do not require any special instrumentation, protein precipitation reagents, extension tubes, phenol/chloroform or protease treatments. Moreover, the kit allows the user to elute into a flexible elution volume ranging from 50 µL to 100 µL. The purified RNA is free from any protein-bound circulating RNA and of the highest integrity. The purified RNA can be used in a number of downstream applications including real time PCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.

Urine Exosome Purification and RNA Isolation Mini Kit

For sample volumes ranging from 250 µL to 1 mL.

Urine Exosome Purification and RNA Isolation Midi Kit

For sample volumes ranging from 2 mL to 10 mL.

Urine Exosome Purification and RNA Isolation Maxi Kit

For sample volumes ranging from 11 mL to 30 mL.

Details

Supporting Data

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Kit Specifications
Minimum Urine Input	2 mL
Maximum Urine Input	10 mL
Size of Exosomes Purified	40 nm – 150 nm
Size of RNA Purified	All sizes, including miRNA and small RNA (< 200 nt)
Elution Volume	50-100 μL
Time to Complete 10 Purifications	35 – 40 minutes
Average Yields*	Variable depending on specimen

*Please check page 4 of the product insert for the average yields and the common RNA quantification methods.

Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.

Important Note
Urine samples stored at -70°C, -20°C or at 4°C will develop some precipitation due to the aggregation of some of the highly abundant proteins in urine. Eliminating these precipitates using centrifugation or filtration may cause the loss of exosomes. Furthermore, these precipitates may affect the quality of the purified nucleic acid. We recommend the use of Norgen’s Urine Preservative when collecting urine samples, which is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures. The components of the Urine Preservative allow samples to be stored for over 2 years at room temperature with no detected degradation of urine DNA, RNA or proteins. Norgen’s Urine Preservative is available as a liquid format in Norgen’s Urine Preservative Single Dose Ampules, as well as in a dried format in Norgen’s Urine Collection and Preservation Tubes.

Component	Cat. 58400 (50 preps)	Cat. 58700 (25 preps)	Cat. 58800 (15 preps)
Slurry E	12.5 mL	12.5 mL	12.5 mL
ExoC Buffer	8 mL	30 mL	50 mL
ExoR Buffer	12 mL	12 mL	12 mL
Lysis Buffer A	20 mL	20 mL	20 mL
Lysis Additive B	2 mL	2 mL	2 mL
Wash Solution A	18 mL	18 mL	18 mL
Elution Solution A	6 mL	6 mL	6 mL
Mini Filter Spin Columns	50	25	15
Mini Spin Columns	50	25	15
Collection Tubes	50	25	15
Elution Tubes (1.7 mL)	100	50	30
Product Insert	1	1	1