Plasma/Serum Cell-Free Circulating DNA Purification Kits

Overview

Isolate all sizes of circulating DNA from plasma and serum samples
Isolate viral and bacterial DNA
Versatile plasma and serum input volumes
Concentrate circulating DNA into a flexible elution volume
Isolate inhibitor-free cell-free circulating DNA
Compatible with Streck Cell-Free DNA BCT® Tubes
Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

These kits provide a fast, reliable and convenient spin column method for the isolation of high quality, high purity and inhibitor-free cell-free circulating DNA (cfc-DNA) from plasma/serum sample volumes. The purified plasma/serum cfc-DNA is fully compatible with all downstream applications including PCR, qPCR, methylation-sensitive PCR and Southern Blot analysis, microarrays and NGS.

Background

Plasma/Serum cell-free circulating DNA (cfc-DNA) has the potential to provide biomarkers for certain cancers and disease states as well as fetal DNA in maternal blood. Currently, significant advancements are being made in utilizing cfc-DNA as biomarkers for the early diagnosis, prognosis and monitoring of therapy for several cancer types and autoimmune diseases. Cell-free mitochondrial DNA (cfmtDNA) is also under investigation for its clinical significance. This cfc-DNA is usually present as short fragments of less than 1000 bp. In addition, cell-free fetal DNA has been widely used as a non-invasive method for prenatal diagnosis including early identification of fetal sex, genetic studies for families at high risk for inherited genetic disorders, screening for Rhesus factor, screening for aneuploidy and identification of preeclampsia.

Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit

Ideal for handling initial plasma/serum volumes ranging from 10 μL up to 200 μL. The kit is designed to isolate all sizes of cfc-DNA from either fresh or frozen plasma/serum samples and the purified DNA is eluted into a flexible elution volume ranging from 25 µL to 50 µL.

Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit

Ideal for handling initial plasma/serum volumes ranging from 200 μL up to 500 μL. The kit is designed to isolate all sizes of cfc-DNA from either fresh or frozen plasma/serum samples and the purified DNA is eluted into a flexible elution volume ranging from 25 µL to 50 µL.

Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit

Ideal for handling initial plasma/serum volumes ranging from 1 mL - 4 mL. The first column will handle the large volume input of bodily fluids that is followed by a concentration on a mini column for a final elution of 25 µL to 50 µL

Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit

Ideal for handling initial plasma/serum volumes ranging from 5 mL- 10 mL. The first column will handle the large volume input of bodily fluids that is followed by a concentration on a mini column for a final elution of 25 µL to 50 µL

Details

Supporting Data

Figure 1. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit was used to purify circulating DNA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant, and compared to Competitor Q's kit. Two microlitres of the purified DNA was then used as the template in qPCR reactions to assess the relative amount of the purified housekeeping 5S rRNA gene. The relative amount of the 5S rRNA gene increases linearly with increasing the sample input volume. Norgen's kit showed the most consistent and the highest recovery of the housekeeping 5S rRNA gene as compared to the other isolation method.

Figure 2. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit as used to purify circulating DNA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified DNA was then used as the template in qPCR reactions to assess the linearity of the purified housekeeping 5S rRNA gene from the different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit was able to recover 96% of the 5S rRNA gene from 100 µL plasma relative to the amount that is present in 50 µL plasma. Moreover, 97% of the 5S rRNA gene was recovered from 200 µL plasma relative to the amount that is present in 100 µL plasma.

Figure 3. DNA was isolated from 50 µL, 100 µL and 200 µL plasma using Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit. Increasing volumes of the elution (2, 4 and 8 μL) were used in a 20 μL qPCR reaction to observe any decrease in Ct value. An increase in Ct values with increasing amount of template would be a clear indication of PCR inhibitors present in the sample. An increase in elution volume used as a template in the qPCR did not affect the Ct value generated from qPCR and in fact the Ct values tend to decrease with increasing the PCR input volume indicating that DNA purified from plasma using Norgen's kit is free of the common inhibitors usually present in plasma.

Figure 4. Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit was used to purify circulating DNA from 200 µL, 300 µL and 400 µL plasma prepared from blood collected on citrate as an anticoagulant in comparison to Competitor Q's kits. Two microlitres of the purified DNA was then used as the template in qPCR reactions to assess the relative amount of the purified housekeeping 5S rRNA gene. The relative amount of the 5S rRNA gene increases linearly with increasing the sample input volume. Norgen's kit showed the most consistent and the highest recovery of the housekeeping 5S rRNA gene as compared to the other isolation method.

Figure 5. Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit was used to purify circulating DNA from 200 µL, 300 µL and 400 µL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified DNA was then used as the template in qPCR reactions to assess the linearity of the purified housekeeping 5S rRNA gene from the different plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit was able to recover 98% of the 5S rRNA gene from 300 µL plasma relative to the amount that is present in 200 µL plasma. Moreover, 98% of the 5S rRNA gene was recovered from 400 µL plasma relative to the amount that is present in 300 µL plasma.

Figure 6. DNA was isolated from 200 µL, 300 µL and 400 µL plasma using Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit. Increasing volumes of the elution (2, 4 and 8 μL) were used in a 20 μL qPCR reaction to observe any decrease in Ct value. An increase in Ct values with increasing amount of template would be a clear indication of PCR inhibitors present in the sample. An increase in elution volume used as a template in the qPCR did not affect the Ct value generated from qPCR and in fact the Ct values tend to decrease with increasing the PCR input volume indicating that DNA purified from plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.

Figure 7. Purification of cell-free circulating DNA from different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit was used to purify circulating DNA from 0.5 mL, 1 mL, 2 mL and 4 mL plasma prepared from blood collected on citrate as an anticoagulant in comparison to Competitor Q's kits. Two microlitres of the purified DNA was then used as the template in qPCR reactions to assess the relative amount of the purified housekeeping 5S rRNA gene. The relative amount of the 5S rRNA gene increases linearly with increasing the sample input volume. Norgen's Kit showed the most consistent and the highest recovery of the housekeeping 5S rRNA gene as compared to the other isolation method.

Figure 8. Linearity of DNA purified from increasing plasma volumes using Norgen’s Plasma/Serum cell-free circulating DNA Purification Midi Kit. Norgen’s Plasma/Serum cell-free circulating DNA Purification Midi Kit was used to purify circulating DNA from 0.5 mL, 1 mL, 2 mL and 4 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified DNA was then used as the template in qPCR reactions to assess the linearity of the purified housekeeping 5S rRNA gene from the different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit was able to recover 97% of the 5S rRNA gene from 1 mL plasma relative to the amount that is present in 0.5 mL plasma. Moreover, 95% of the 5S rRNA gene was recovered from 2 mL plasma relative to the amount that is present in 1 mL plasma. Additionally, 95% of the 5S rRNA gene was recovered from 4 mL plasma relative to the amount that is present in 2 mL plasma.

Figure 9. Determination of the amount of inhibition present in plasma cell-free circulating DNA samples when detecting the human 5S gene. DNA was isolated from 0.5 mL, 1 mL, 2 mL and 4 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit. Increasing volumes of the elution (2, 4 and 8 µL) were used in a 20 µL qPCR reaction to observe any decrease in Ct value. An increase in Ct values with increasing amount of template would be a clear indication of PCR inhibitors present in the sample. An increase in elution volume used as a template in the qPCR did not affect the Ct value generated from qPCR, and in fact the Ct values tend to decrease with increasing the PCR input volume indicating that DNA purified from plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.

Figure 10. Norgen's Plasma/Serum cell-free circulating DNA Purification Maxi Kit was used to purify circulating DNA from 5 mL, 8 mL and 10 mL plasma prepared from blood collected on citrate as an anticoagulant in comparison to Competitor Q's kits. Two milliliters of the purified DNA was then used as the template in qPCR reactions to assess the relative amount of the purified housekeeping 5S rRNA gene. The relative amount of the 5S rRNA gene increases linearly with increasing the sample input volume. Norgen's kit showed the most consistent and the highest recovery of the housekeeping 5S rRNA gene as compared to the other isolation method.

Figure 11. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit was used to purify circulating DNA from 5 mL, 8 mL and 10 mL plasma prepared from blood collected on citrate as an anticoagulant. Two milliliters of the purified DNA was then used as the template in qPCR reactions to assess the linearity of the purified housekeeping 5S rRNA gene from the different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit was able to recover 94% of the 5S rRNA gene from 8 mL plasma relative to the amount that is present in 5 mL plasma. Moreover, 96% of the 5S rRNA gene was recovered from 10 mL plasma relative to the amount that is present in 10 mL plasma.

Figure 12. DNA was isolated from 5 mL, 8 mL and 10 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit. Increasing volumes of the elution (2, 4 and 8 µL) were used in a 20 µL qPCR reaction to observe any decrease in Ct value. An increase in Ct values with increasing amount of template would be a clear indication of PCR inhibitors present in the sample. An increase in elution volume used as a template in the qPCR did not affect the Ct value generated from qPCR and in fact the Ct values tend to decrease with increasing the PCR input volume indicating that DNA purified from plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.

Kit Specifications
Minimum Plasma/Serum Input	10 μL
Maximum Plasma/Serum Input	200 μL
Size of DNA Purified	≥ 50 bp
Elution Volume	25-50 μL
Time to Complete 10 Purifications	15-20 minutes
Average Yields*	Variable depending on specimen

*Please check page 7 in the product manual for the Plasma/Serum Average Yields and the Common DNA Quantification Methods.

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. The kit contains a ready-to-use Proteinase K, which is dissolved in a specially prepared storage buffer. The buffered Proteinase K is stable for up to 2 years after the date of shipment when stored at room temperature.

Component	Cat. 55500 (50 preps)	Cat. 55100 (50 preps)	Cat. 55600 (20 preps)	Cat. 55800 (10 preps)
Binding Buffer B	40 mL	2 x 40 mL	2 x 85 mL 1 x 12 mL	2 x 85 mL 1 x 40 mL
Proteinase K	0.6 mL	2 x 1 mL	6.5 mL	8 mL
Solution WN	18 mL	18 mL	18 mL	18 mL
Wash Solution A	18 mL	38 mL	18 mL	18 mL
Elution Buffer B	8 mL	8 mL	30 mL	30 mL
Micro Spin Columns	50	50	20	10
Mini Spin Columns	-	50	-	-
Midi Spin Columns	-	-	20	-
Maxi Spin Columns	-	-	-	10
Collection Tubes	50	100	20	10
Elution Tubes (1.7 mL)	50	100	20	10
Product Insert	1	1	1	1

Documentation

RELATED BLOGS

FAQs

Micro, Mini, Midi, Maxi

What if a variable speed centrifuge is not available and the speed differs from the recommended?

A fixed speed centrifuge can be used, however reduced yields may be observed.

At what temperature should I centrifuge my samples?

All centrifugation steps are performed at room temperature. Centrifugation at 4 degrees Celsius will not adversely affect kit performance.

What if I added more or less of the specified reagents’ volume?

Adding more or less than the specified volumes may reduce both the quality and the quantity of the purified DNA. Eluting your DNA in high volumes will increase the yield but will lower the concentration. Eluting in small volumes will increase the concentration but will lower the overall yield.

What If I forgot to do a dry spin before my final elution step?

Your purified DNA will be contaminated with the Wash Solution A. This may reduce the quality of your purified DNA and will interfere with your downstream applications.

Can I perform a second elution?

Yes, but it is recommended that the 2nd elution be in a smaller volume (50% of 1st Elution). It is also recommended to perform the 2nd elution into a separate elution tube to avoid diluting the 1st elution.

What if my incubation time varied from what is specified in the product manual?

Varying the incubation time will result in a reduction in your DNA yields.

Why do my samples show very low DNA yield?

Plasma/Serum samples contain very little cfc-DNA. This varies from individual to individual. In order to increase the yield, the amount of Plasma/Serum input could be increased.

Why does my purified cfc-DNA not perform well in downstream applications?

If a different Elution Buffer was used other than the one provided in the kit, the buffer should be checked for any components that may interfere with the application. Common components that are known to interfere are high salts (including EDTA), detergents and other denaturants. Check the compatibility of your Elution Buffer with the intended use.

Citations

Micro (55500)

Title	Plasma cfDNA abundance as a prognostic biomarker for higher risk of death in geriatric cardiovascular patients
Journal	Mechanisms of Ageing and Development. 2024.
Authors	YMaurizio Cardelli a, Francesca Marchegiani b, Pierpaolo Stripoli b, Francesco Piacenza a, Rina Recchioni b, Mirko Di Rosa c, Robertina Giacconi a, Marco Malavolta a, Roberta Galeazzi b, Beatrice Arosio d e, Fiammetta Cafarelli f, Francesco Spannella g h, Antonio Cherubini i, Fabrizia Lattanzio j, Fabiola Olivieri

Mini (55100)

Title	Cell-free fetal DNA as a non-invasive method using pyrosequencing in detecting beta-globin gene mutation: A pilot study from area with limited facilities in Indonesia
Journal	Front. Pediatr. 2022.
Authors	Ani Melani Maskoen, Nurul Setia Rahayu, Bremmy Laksono, Azzania Fibriani, Willyanti Soewondo, Johanes C. Mose, Edhyana Sahiratmadja and Ramdan Panigoro

Title	Early detection of colorectal cancer somatic mutations using cfDNA liquid biopsies in a murine carcinogenesis model
Journal	Journal of Cancer. 2022.
Authors	Héctor Martínez-Gregorio, Clara Estela Díaz-Velásquez, Mario Esaú Romero-Piña, Miguel Ruiz De La Cruz, Norma Laura Delgado-Buenrostro, Aldo De La Cruz-Montoya, Yolanda Irasema Chirino, Luis Ignacio Terrazas, Luis Alberto Medina, Felipe Vaca-Paniagua

Title	Chronic Inhibition of Toll‐Like Receptor 9 Ameliorates Pulmonary Hypertension in Rats
Journal	Journal of the American Heart Association. 2021.
Authors	Tomohito Ishikawa, Kohtaro Abe, Mariko Takana‐Ishikawa, Keimei Yoshida, Takanori Watanabe, Satomi Imakiire, Kazuya Hosokawa, Mayumi Hirano, Katsuya Hirano and Hiroyuki Tsutsui

Title	A multiparametric extraction method for Vn96-isolated plasma extracellular vesicles and cell-free DNA that enables multi-omic profiling
Journal	Scientific Reports. 2021.
Authors	Jeremy W. Roy, Catherine A. Taylor, Annie P. Beauregard, Surendar R. Dhadi, D. Craig Ayre, Sheena Fry, Simi Chacko, Gabriel Wajnberg, Andrew P. Joy, Ngoc-Nu Mai-Thi, Nicolas Crapoulet, David A. Barnett, Anirban Ghosh, Stephen M. Lewis & Rodney J. Ouellette

Title	554P KRAS-dependent and independent mechanisms of progressive disease (PD) in colorectal cancer (CRC) patients (pts) with liver metastases (LM) while monitoring on circulating cell free DNA (cfDNA)
Journal	Annals of Oncology. October 23, 2018.
Authors	A Kirov Z Mihaylova, V Petrova, T Todorov, D Petkova, A Garev, A Todorova-Georgieva

Title	Advances in Circulating Tumor DNA Analysis
Journal	Advances in Clinical Chemistry. 2017.
Authors	S. Perakis, M. Auer, J. Belic, E. Heitzer

Title	Comprehensive evaluation of methods to isolate, quantify, and characterize circulating cell-free DNA from small volumes of plasma
Journal	Analytical and Bioanalytical Chemistry. 2015.
Authors	Mauger F, Dulary C, Daviaud C, Deleuze JF, Tost J.

Plasma/Serum Cell-Free Circulating DNA Purification Kits