Plasma/Serum Cell-Free Circulating DNA Purification Kits Dx

Features and Benefits

Concentrate circulating DNA into a flexible elution volume ranging from (25 µL - 50 µL)
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
Research use format is also available

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. These kits are 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. 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 mitocondrial 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 Dx

For serum input volumes 10 µL - 200 µL. Purify high-quality DNA in 15-20 minutes

Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit Dx

For serum input volumes 200 µL - 500 µL. Purify high-quality DNA in 15-20 minutes.

Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit Dx

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. All components for the purification & concentration are provided in one convenient & fast kit for the easy processing of large input volumes of bodily fluids. For a schematic workflow of the protocol click here to view. For serum input volumes 1 mL - 4 mL. Purify high-quality DNA in 90 minutes.

Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit Dx

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. All components for the purification & concentration are provided in one convenient & fast kit for the easy processing of large input volumes of bodily fluids. For a schematic workflow of the protocol click here to view. For serum input volumes 5 mL - 10 mL. Two kits in one - purify and concentrate DNA from bodily fluids using a convenient two column system.

Details

Supporting Data

Figure 1. 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 2. 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 3. 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 4. 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 5. 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 6. 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.

Figure 7. 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 8. 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 9. 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 10. 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 11. 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 12. 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.

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 buffers should be kept tightly sealed and stored at room temperature (15-25°C) for up to 2 years without showing any reduction in performance. Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Kits contain ready-to-use Proteinase K solution, which is dissolved in a specially prepared storage buffer. The Proteinase K is stable for up to 2.5 years after delivery when stored at room temperature. To prolong the lifetime of Proteinase K, storage at 2–8°C is recommended.

Component	Cat. Dx55100 (50 preps)	Cat. Dx55500 (50 preps)	Cat. Dx55600 (20 preps)	Cat. Dx55800 (10 preps)
Binding Buffer B	2 x 40 mL	40 mL	2 x 85 mL 1 x 12 mL	2 x 85 mL 1 x 40 mL
Proteinase K	2 x 1 mL	0.6 mL	6.5 mL	8 mL
Solution WN	18 mL	18 mL	18 mL	18 mL
Wash Solution A	38 mL	18 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	100	50	20	10
Elution Tubes (1.7 mL)	100	50	20	10
Product Insert	1	1	1	1

Documentation

RELATED BLOGS

Citations

Micro (Dx55500)

Title	Evaluation of commercial kits for purification of circulating free DNA
Journal	Cancer Genetics. 2019.
Authors	Russell J. Diefenbach, Jenny H. Lee, Richard F. Kefford, Helen Rizos,

Title	Enzyme-Powered Three-Dimensional DNA Nanomachine for DNA Walking, Payload Release, and Biosensing
Journal	ACS Nano. 2016.
Authors	Yang, X., Tang, Y., Mason, S. D., Chen, J., & Li, F

Mini (Dx55100)

Title	Evaluation of commercial kits for purification of circulating free DNA
Journal	Cancer Genetics. 2019.
Authors	Russell J. Diefenbach, Jenny H. Lee, Richard F. Kefford, Helen Rizos,

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.

Midi (Cat. Dx55600)

Title	Evaluation of commercial kits for purification of circulating free DNA
Journal	Cancer Genetics. 2019.
Authors	Russell J. Diefenbach, Jenny H. Lee, Richard F. Kefford, Helen Rizos,

Maxi kit (Dx55800)

Title	Evaluation of commercial kits for purification of circulating free DNA
Journal	Cancer Genetics. 2019.
Authors	Russell J. Diefenbach, Jenny H. Lee, Richard F. Kefford, Helen Rizos,

Plasma/Serum Cell-Free Circulating DNA Purification Kits Dx