Multiplex I cfDNA Reference Standard Set in Synthetic Plasma

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. Multiplex I cfDNA Reference Standard Set in Synthetic Matrix contains 8 onco-relevant mutations to measure your extraction efficiency.

This product is designed to be compatible with several commonly used extraction kits. Please see the supporting data tab for full details. Customers using Thermo MagMAX or Machery Nagel Nucleosnap extraction platforms are encouraged to use our alternative product - Multiplex I cfDNA in Synthetic Matrix II.

Extraction Platform Capability

Cat no.	Description	Extraction platform
		Column based		Column/Bead	Bead based
		QiAamp Circulating Nucleic Acid Kit	Macherey Nagel Nucleosnap Plasma Kit	Qiagen Min Elute cfDNA Kit	MagMax cfDNA Isolation Kit	Maxwell RSC cfDNA Isolation Kit	Chemagen cfDNA Extraction Kit
HD816	Multiplex I cfDNA in synthetic plasma	✓	Not recommended	✓	Not recommended	✓	Not recommended
HD817	Multiplex I cfDNA in synthetic matrix	✓	Not recommended	✓	Not recommended	✓	Not recommended
HD917	Multiplex I cfDNA in synthetic matrix II	✓	✓	✓	✓	✓	✓

The Multiplex I cfDNA Reference Standard Set in synthetic plasma 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

Cosmics: 1,500+

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

Buffer: Synthetic plasma

Expected DNA yield: ≥240 ng DNA (from 2 ml of plasma) using the QIAamp® Circulating Nucleic Acid Kit

Product information

Fragment size: 170bp

Unit size: 400ng per 1ml (8 vials total)

Verified mutations

100% Multiplex I Wild Type cfDNA Reference Standard (Part No.: HD815)

Chromosome	Gene	Variant	Expected Allelic Frequency
7p12	EGFR	L858R	0.00%
7p12	EGFR	ΔE746 - A750	0.00%
7p12	EGFR	T790M	0.00%
7p12	EGFR	A767_V769dup	0.00%
12p12.1	KRAS	G12D	0.00%
1p13.2	NRAS	Q61K	0.00%
1p13.2	NRAS	A59T	0.00%
3q26.3	PIK3CA	E545K	0.00%

5% Multiplex I cfDNA Reference Standard (Part No.: HD812)

Chromosome	Gene	Variant	Expected Allelic Frequency
7p12	EGFR	L858R	5.0%
7p12	EGFR	ΔE746 - A750	5.0%
7p12	EGFR	T790M	5.0%
7p12	EGFR	A767_V769dup	5.0%
12p12.1	KRAS	G12D	6.3%
1p13.2	NRAS	Q61K	6.3%
1p13.2	NRAS	A59T	6.3%
3q26.3	PIK3CA	E545K	6.3%

1% Multiplex I cfDNA Reference Standard (Part No.: HD813)

Chromosome	Gene	Variant	Expected Allelic Frequency
7p12	EGFR	L858R	1.0%
7p12	EGFR	ΔE746 - A750	1.0%
7p12	EGFR	T790M	1.0%
7p12	EGFR	1.0%
12p12.1	KRAS	G12D	1.3%
1p13.2	NRAS	Q61K	1.3%
1p13.2	NRAS	A59T	1.3%
3q26.3	PIK3CA	E545K	1.3%

0.1% Multiplex I cfDNA Reference Standard (Part No.: HD814)

Chromosome	Gene	Variant	Expected Allelic Frequency
7p12	EGFR	L858R	0.10%
7p12	EGFR	ΔE746 - A750	0.10%
7p12	EGFR	T790M	0.10%
7p12	EGFR	A767_V769dup	0.10%
12p12.1	KRAS	G12D	0.13%
1p13.2	NRAS	Q61K	0.13%
1p13.2	NRAS	A59T	0.13%
3q26.3	PIK3CA	E545K	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.

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

General information

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)

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.