Mimix™ Multiplex I, cfDNA in Synthetic Matrix II Reference Standard

The most flexible reference standard for cfDNA recovery

HD917 is the replacement for and an optimized version of HD816 - Multiplex I cfDNA Reference Standard Set in Synthetic Plasma and HD817 - Multiplex I cfDNA Reference Standard Set in Synthetic Matrix. It is designed to be compatible with the most commonly used extraction kits - including bead and column based methods (see supporting data) - making it the most flexible reference standard for cfDNA recovery.

Multiplex I cfDNA Reference Standard Set in Synthetic Matrix II (HD917) contains 8 onco-relevant mutations to measure your extraction efficiency and assay performance. As an optimal synthetic matrix that enables extraction recovery of cfDNA by multiple purification methods, its clinically relevant mutations in an artificial matrix give you full control of your cfDNA workflow, reducing the likelihood of a false negative or positive due to incorrect assay loading.

The Multiplex I cfDNA Reference Standard Set in artificial matrix covers multiple engineered single nucleotide variants (SNVs/SNPs) with 8 mutations at 5%, 1%, and 0.1% allelic frequencies.

For information on specific gene copies detected in each product batch, download the product insert from the resources tab.

Technical data

Format: cfDNA

Genes covered: EGFR, KRAS, NRAS, PIK3CA

Allelic frequencies: 5%, 1%, 0.1% and 0% (100% wild type)

Buffer: Synthetic matrix II

Expected DNA yield: ≥120 ng DNA per mL of product using the QIAamp® Circulating Nucleic Acid Kit with 2 mL extraction input

Product information

Fragment size: 170bp

Unit size: 400ng per 1ml (4 vials total)

Extraction platform compatibility

Product	Extraction platform
	Column based		Column/Bead	Bead Based
	QIAamp Circulating Nucleic Acid Kit	Macherey Nagel NucleoSnap Plasma kit	QIAgen MinElute cfDNA kit	MagMAX cfDNA isolation kit	Maxwell RSC cfDNA isolation kit
Synthetic Matrix II

Verified mutations

HD917 component expected allelic frequencies (%)

Gene	Variant (AA)	CDS mutation	GRCh38 co-ordinates	HD912 5%	HD913 1%	HD914 0.1%
EGFR	T790M	c.2369C>T	7:55181378	5	1	0.1
EGFR	delE746-A750	c.2235_2249del	7:55174772-55174786	5	1	0.1
EGFR	L858R	c.2573T>G	7:55191822	5	1	0.1
EGFR	A767_V769dup	c.2300_2308dup	7:55181317-55181318	5	1	0.1
NRAS	Q61K	c.181C>A	1:114713909	6.3	1.3	0.13
NRAS	A59T	c.175G>A	1:114713915	6.3	1.3	0.13
PIK3CA	E545K	c.1633G>A	3:179218303	6.3	1.3	0.13
KRAS	G12D	c.35G>A	12:25245350	6.3	1.3	0.13

Presence confirmed in parental cell line: EGFR G719S (20% allelic frequency)

Exome sequencing data: The cfDNA in buffer format of this product (HD780) has been verified by whole exome sequencing.

General information

Shipping conditions: Ambient (shipped with gel packs to ensure item maintains room temperature storage during transport)

Storage: 4°C

Expiration: See all product shelf life information

Quality control

Fragmentation size: D1000 DNA ScreenTape assay

Allelic frequency: Droplet Digital™ PCR

Quantification: Qubit dsDNA BR Assay (post-fragmentation)

Intended use: For assay developers and molecular diagnostic labs for routine performance monitoring of molecular biology assays. Research use only. Not for diagnostic procedures.

We have summarized the extraction efficiency of Matrix I (HD816/HD817) and Matrix II (HD917) using a range of the most used extraction kits.

Introduction

The analysis of cell-free circulating nucleic acids is becoming a powerful tool for the provision of personalized medicine to cancer patients. Clinical samples have extremely low quantities of cfDNA so it is important to ensure that cell-free DNA extraction from plasma is as efficient as possible. Cell-free DNA reference standards allow us to monitor the performance of extraction techniques, as well as downstream molecular assays. The use of a synthetic plasma to make reference standards is often preferred over human plasma. This is due to its stability, lot-to-lot consistency, and absence of interfering substances. Not all synthetic plasmas are compatible with the existing range of extraction methods and molecular assays. Here we demonstrate the compatibility of a cell-free DNA reference standard in synthetic plasma with a range of different extraction techniques, including the two most popular approaches: column-based extraction and magnetic bead-based extraction.

Method

Our Matrix I (HD817) and Matrix II (HD917) products consist of cfDNA containing known variants at specific allelic frequencies spiked into synthetic plasmas. The spiked-in cfDNA was extracted from Matrix I and Matrix II using five popular cfDNA extraction kits, which include the two most used extraction technologies: bead based and column-based chemistry.

Extraction Kits used in this study:
ThermoFisher MagMAX™ Cell-Free DNA Isolation Kit (#A29319)
Qiagen MinElute ccfDNA Kit (#52204)
Qiagen QIAamp Circulating Nucleic Acid Kit (#55114)
Macherey Nagel NucleoSnap DNA Plasma (#740300)
Maxwell RSC ccfDNA Plasma Kit (Cat #AS1480)

Extraction efficiencies with 5 cfDNA extraction kits

Result

Matrix I (HD816/HD817) demonstrated satisfactory efficiencies in three of the kits tested, however a low yield is recovered when using Machery Nagel Nucleosnap kit, and no cfDNA is recovered using the ThermoFisher MagMAX extraction kit. On the other hand, Matrix II (HD917) demonstrated higher efficiencies and compatibility with the most common extraction kits used for cfDNA isolation, including MagMAX and Machery Nagel Nucleosnap.