Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Kits

Overview

Isolate all sizes of circulating DNA, circulating and exosomal RNA, including microRNA, viral DNA/RNA in one elution
Versatile plasma/serum input ranges
No phenol extractions
No carrier RNA
Bind and elute all RNA irrespective of size or GC content, without bias
Concentrate circulating DNA, circulating and exosomal RNA, viral DNA and viral RNA into flexible elution volumes
Compatible with Streck Cell-Free DNA BCT® Tubes
Purified RNA is suitable for a variety of downstream applications, including Small RNA Sequencing. Find out more information on Norgen's NGS services

These kits provide a fast, reliable and convenient method to purify and concentrate high quality, high purity and inhibitor-free cell-free circulating DNA, circulating and exosomal RNA, as well as viral DNA/RNA from fresh or frozen serum or plasma samples prepared from blood collected on either EDTA or Citrate. The purified plasma/serum circulating and viral nucleic acid is fully compatible with all downstream applications including PCR, qPCR, methylation-sensitive reverse transcription qPCR, reverse transcription PCR, methylation-sensitive PCR and Southern Blot analysis, Northern blotting, RNase protection and primer extension, expression array assays, and NGS. Plasma samples prepared from blood collected on heparin should not be used as heparin can significantly interfere with many downstream applications such as RT-PCR.

Background

Plasma/serum cell-free circulating and viral nucleic acid has the potential to provide biomarkers for certain cancers and disease states as well as for the detection of viral infection. 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 Exosomes are 40 - 150 nm membrane vesicles, which are secreted by most cell types. Exosomes can be found in saliva, blood, urine, amniotic fluid and malignant ascitic fluids, among other biological fluids. Evidence has been accumulating recently that these vesicles act as cellular messengers, conveying information to distant cells and tissues within the body. These exosomes may play a functional role in mediating adaptive immune responses to infectious agents and tumours, tissue repair, neural communication and transfer of pathogenic proteins. For this reason exosomal RNAs may serve as biomarkers for various diseases including cancer. As the RNA molecules encapsulated within exosomes are protected from degradation by RNAses they can be efficiently recovered from biological fluids, such as plasma or serum.

Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit

For the easy processing of small input volumes ranging from 50 µL to 200 µL.

Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit

For input volumes ranging from 250 µL to 1.5 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 50 µL to 100 µL.

Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit

For input volumes ranging from 2 mL to 5 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 50 µL to 100 µL.

Details

Supporting Data

Figure 1. Purification of cell-free circulating DNA from different plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300) was 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 NA was then used as the template in qPCR reactions to assess the relative amount of the purified the housekeeping 5S rRNA gene. The Ct values for the 5S rRNA gene is linearly decreasing with increasing the sample input volume.

Figure 2. Linearity of DNA purified from increasing plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300) was used to purify circulating NA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the linearity of the purified the housekeeping 5S rRNA gene from the different plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit was able to recover ninety-four percent of the 5S rRNA gene from 100 µL plasma relative to the amount that is present in 50 µL plasma. Moreover, 98% of the 5S rRNA gene was recovered from 200 µL plasma relative to the amount that is present in 100 µL plasma.

Figure 3. Determination of the amount of inhibition present in plasma cell-free circulating DNA samples when detecting the human 5S gene. DNA was isolated from 50 µL, 100 µL and 200 µL plasma using Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300). 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. Purification of cell-free circulating RNA and exosomal RNA from different plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300) was used to purify cell-free circulating and exosomal RNA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant in comparison. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of the purified (A) housekeeping 5S rRNA transcript and (B) miR-21. The average Ct value for both (A) 5S rRNA transcript and (B) miR-21 is linearly decreasing with increasing the sample input volume.

Figure 5. Linearity of RNA purified from increasing plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300) was used to purify RNA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the linearity of the purified (A) housekeeping 5S rRNA transcript and (B) miR-21 from the different plasma volumes. Norgen's Plasma/Serum Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit was able to recover 91% of the 5S rRNA transcript and 95% of miR-21 from 100 µL plasma relative to the amount that is present in 50 µL plasma. Moreover, 90% of the 5S rRNA transcript and 92% of the miR-21 was recovered from 200 µL plasma relative to the amount that is present in 100 µL plasma.

Figure 6. Determination of the amount of inhibition present in plasma RNA samples when detecting the human 5S transcript and miR-21. RNA was isolated from 50 µL, 100 µL and 200 µL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300). Increasing volumes of the elution (2, 4 and 8 µL) were used in a 20 µL reverse transcription reaction followed by qPCR amplification 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 the PCR input volume used as a template in the reverse transcription reaction did not affect the Ct value generated from the qPCR amplification for both (A) 5S rRNA transcript and (B) miR-21. In fact the Ct values tend to decrease with increasing the PCR input volume indicating that RNA 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 and Viral Nucleic Acid Purification Midi Kit (Cat# 56400) was used to purify circulating DNA from 250 µL, 750 µL and 1.5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the relative amount of the purified the housekeeping 5S rRNA gene. The Ct values for the 5S rRNA gene are linearly decreasing with increasing the sample input volume.

Figure 8. Linearity of DNA purified from increasing plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400) was used to purify circulating NA from 250 µL, 750 µL and 1.5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the linearity of the purified the housekeeping 5S rRNA gene from the different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit was able to recover 98% of the 5S rRNA gene from 750 µL plasma relative to the amount that is present in 250 µL plasma. Moreover, 93% of the 5S rRNA gene was recovered from 1.5 mL plasma relative to the amount that is present in 750 µL 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 250 µL, 750 µL and 1.5 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400). 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 RNA and exosomal RNA from different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400) was used to purify cell-free circulating and exosomal RNA from 250 µL, 750 µL and 1.5 mL plasma prepared from blood collected on citrate as an anticoagulant in comparison. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of the purified (A) housekeeping 5S rRNA transcript and (B) miR-21. The average Ct value for both (A) 5S rRNA transcript and (B) miR-21 is linearly decreasing with increasing the sample input volume.

Figure 11. Linearity of RNA purified from increasing plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400) was used to purify RNA from 250 µL, 750 µL and 1.5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the linearity of the purified (A) housekeeping 5S rRNA transcript and (B) miR-21 from the different plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit was able to recover 97% of both the 5S rRNA transcript and miR-21 from 750 µL plasma relative to the amount that is present in 250 µL plasma. Moreover, ninety-four percent of the 5S rRNA transcript and 97% of the miR-21 was recovered from 1.5 mL plasma relative to the amount that is present in 750 µL plasma.

Figure 12. Determination of the amount of inhibition present in plasma RNA samples when detecting the human 5S transcript and mir-21. RNA was isolated from 250 µL, 750 µL and 1.5 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400). Increasing volumes of the elution (2, 4 and 8 µL) were used in a 20 µL reverse transcription reaction followed by qPCR amplification 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 the PCR input volume used as a template in the reverse transcription reaction did not affect the Ct value generated from the qPCR amplification for both (A) 5S rRNA transcript and (B) miR-21. In fact the Ct values tend to decrease with increasing the PCR input volume indicating that RNA purified from plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.

Figure 13. Purification of cell-free circulating DNA from different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500) was used to purify circulating DNA from 2 mL, 3 mL and 5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the relative amount of the purified the housekeeping 5S rRNA gene. The Ct. values for the 5S rRNA gene is linearly decreasing with increasing the sample input volume.

Figure 14. Linearity of DNA purified from increasing plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500) was used to purify circulating NA from 2 mL, 3 mL and 5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the linearity of the purified the housekeeping 5S rRNA gene from the different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit was able to recover 98% of the 5S rRNA gene from 3 mL plasma relative to the amount that is present in 2 mL plasma. Moreover, 93% of the 5S rRNA gene was recovered from 5 mL plasma relative to the amount that is present in 3 mL plasma.

Figure 15. Determination of the amount of inhibition present in plasma cell-free circulating DNA samples when detecting the human 5S gene. DNA was isolated from 2 mL, 3 mL and 5 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500). 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 16. Purification of cell-free circulating RNA and exosomal RNA from different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500) was used to purify cell-free circulating and exosomal RNA from 2 mL, 3 mL and 5 mL plasma prepared from blood collected on citrate as an anticoagulant in comparison. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of the purified (A) housekeeping 5S rRNA transcript and (B) miR-21. The average Ct value for both (A) 5S rRNA transcript and (B) miR-21 is linearly decreasing with increasing the sample input volume.

Figure 17. Linearity of RNA purified from increasing plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500) was used to purify RNA from 2 mL, 3 mL and 5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the linearity of the purified (A) housekeeping 5S rRNA transcript and (B) miR-21 from the different plasma volumes. Norgen’s Plasma/Serum Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit was able to recover 97% of both the 5S rRNA transcript and miR-21 from 3 mL plasma relative to the amount that is present in 2 mL plasma. Moreover, 94% of the 5S rRNA transcript and 97% of the miR-21 was recovered from 5 mL plasma relative to the amount that is present in 3 mL plasma.

Figure 18. Determination of the amount of inhibition present in plasma RNA samples when detecting the human 5S transcript and mir-21. RNA was isolated from 2 mL, 3 mL and 5 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500). Increasing volumes of the elution (2, 4 and 8 μL) were used in a 20 μL reverse transcription reaction followed by qPCR amplification 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 the PCR input volume used as a template in the reverse transcription reaction did not affect the Ct value generated from the qPCR amplification for both (A) 5S rRNA transcript and (B) miR-21. In fact the Ct values tend to decrease with increasing the PCR input volume indicating that RNA purified from plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.

Kit Specifications
Sample Type	Plasma/Serum
Anti-Coagulant (for Plasma)*	EDTA or Citrate
Sample Volume Range	50 - 200 μL
Size of RNA Purified	All sizes including miRNA and small RNA (< 200 nt)
Size of DNA Purified	≥ 50 bp
Minimum Elution Volume	10 μL
Maximum Elution Volume	25 μL
Time to Complete 10 Purifications	15-20 minutes
Average Yield**	Variable depending on specimen

^{*This kit is suitable for the isolation of total nucleic acid (RNA and DNA) from fresh or frozen serum or plasma prepared from blood collected on either EDTA or Citrate. Plasma samples prepared from blood collected on heparin should not be used, especially if the primarily interest is RNA, as heparin can significantly interfere with many downstream applications such as RT-PCR. If the main interest is DNA then heparinised plasma can be used.} ^{**Please check page 7 of the product insert for average plasma/serum yields and common RNA/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. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.

Component	Cat. 56300 (50 preps)	Cat. 56400 (20 preps)	Cat. 56500 (10 preps)
Lysis Buffer A	2 x 20 mL	100 mL	1 x 130 mL 1 x 30 mL
Wash Solution A	18 mL	1 x 38 mL 1 x 18mL	38 mL
Elution Buffer F	6 mL	15 mL	15 mL
Micro Spin Columns	50	-	-
Mini Spin Columns	-	20	10
Midi Spin Columns	-	20	-
Maxi Spin Columns	-	-	10
Collection Tubes	50	20	10
Elution Tubes (1.7 mL)	50	20	10
Product Insert	1	1	1

Documentation

Citations

Mini (56300)

Title	Diagnostic Study on NSP5 of Human Rotavirus in Najaf Governorate
Journal	Kufa Journal for Nursing Sciences. 2015.
Authors	Saif Jabbar Yasir

Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Kits