Urine Cell-Free Circulating and Viral Nucleic Acid Purification Kits

Features and Benefits

Isolate all sizes of circulating DNA, circulating and exosomal RNA, including microRNA, viral DNA/RNA in one elution
Versatile urine input ranges
No phenol extractions nor carrier RNA
Bind and elute all RNA irrespective of size or GC content, without bias
Concentrate circulating DNA, circulating RNA and exosomal RNA, viral DNA, viral RNA into a flexible elution volumes
Compatible with fresh, frozen or preserved urine sample
Purified RNA is suitable for a variety of downstream applications, including Small RNA Sequencing. Find out more information on Norgen's NGS services
Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

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 RNA including exosomal RNA as well as viral DNA/RNA from fresh, frozen or preserved urine samples. Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix. These kits are designed to isolate all sizes of cfc-DNA and cfc-RNA, including microRNA, as well as all sizes of exosomal RNA. Norgen’s Urine Cell-Free Circulating and Viral Nucleic Acid Purification Kits provide a clear advantage over other available kits in that they do not require phenol/chloroform or any protease treatments. Moreover, the kits allow the user to elute into a flexible elution volume ranging from 50 µL to 100 µL. The purified nucleic Acid is of the highest integrity, and can be used in a any 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.

Background

Recent evidence indicates that cell-free circulating (cfc) nucleic acids such as DNA and RNA, including exosomal RNA, in urine contains valuable information for the discovery of biomarkers that can help with early detection of certain cancer types, for monitoring the disease status as well as for the detection fetal DNA. Moreover, urine has been widely used for the detection of viral infections, including HIV, HCV and HBV.The advantage for using urine as a source for cancer biomarkers is that it can be acquired in large quantities without using invasive procedures, and repeated sampling from the same individual is applicable, which facilitates longitudinal studies. There are many advantages favouring the use of urinary nucleic acids for cancer biomarker discovery over blood, tissue samples or other bodily fluids, including: (1) urine is non-infectious for HIV and less infectious for many other pathogens; (2) the profile of urinary nucleic acids is similar to that in urine or serum; (3) nucleic acid purification from urine is technically much easier because of its low protein concentration (1000-fold lower than blood).

Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit

For sample input volumes ranging from 250 µL to 2 mL.

Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit

For sample input volumes ranging from 2 mL - 10 mL.

Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit

For sample input volumes ranging from 10 mL - 30 mL.

Details

Supporting Data

Figure 1. Purification of cell-free circulating DNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 59900) was used to purify circulating DNA from 500 µL, 1 mL and 2 mL of urine. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the relative amount of the purified housekeeping 5S rRNA gene. The Ct values for the 5S rRNA gene are linearly decreasing with increasing the sample input volume.

Figure 2. Linearity of DNA purified from increasing urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 59900) was used to purify circulating NA from 500 µL, 1 mL and 2 mL urine. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the linearity of the purified housekeeping 5S rRNA gene from the different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit was able to recover 91% of the 5S rRNA gene from 1 mL urine relative to the amount that is present in 500 µL urine. Moreover, 95% of the 5S rRNA gene was recovered from 2 mL urine relative to the amount that is present in 1 mL urine.

Figure 3. Determination of the amount of inhibition present in urine cell-free circulating DNA samples when detecting the human 5S gene. DNA was isolated from 500 µL, 1 mL and 2 mL urine using Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 59900). 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. In fact, the Ct values tend to decrease with increasing the PCR input volume indicating that DNA purified from urine using Norgen's kit is free of the common inhibitors usually present in urine.

Figure 4. Purification of cell-free circulating RNA and exosomal RNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 59900) was used to purify cell-free circulating and exosomal RNA from 500 µL, 1 mL and 2 mL urine. 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 urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 59900) was used to purify RNA from 500 µL, 1 mL and 2 mL urine. 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 urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit was able to recover 92% of the 5S rRNA transcript and 95% of miR-21 from 1 mL urine relative to the amount that is present in 500 µL urine. Moreover, 92% of the 5S rRNA transcript and 92% of the miR-21 was recovered from 2 mL urine relative to the amount that is present in 1 mL urine.

Figure 6. Determination of the amount of inhibition present in urine RNA samples when detecting the human 5S transcript and miR-21. RNA was isolated from 500 µL, 1 mL and 2 mL urine using Norgen’s Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 59900). 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 the (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 urine using Norgen’s kit is free of the common inhibitors usually present in urine.

Figure 7. Purification of cell-free circulating DNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000) was used to purify circulating DNA from 2 mL, 5 mL and 10 mL Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the relative amount of the purified 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 urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000) was used to purify circulating NA from 2 mL, 5 mL and 10 mL urine. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the linearity of the purified housekeeping 5S rRNA gene from the different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit was able to recover 92% of the 5S rRNA gene from 5 mL urine relative to the amount that is present in 2 mL urine. Moreover, 99% of the 5S rRNA gene was recovered from 10 mL urine relative to the amount that is present in 5 mL urine.

Figure 9. Determination of the amount of inhibition present in urine cell-free circulating DNA samples when detecting the human 5S gene. DNA was isolated from 2 mL, 5 mL and 10 mL urine using Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000). 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. In fact the Ct values tend to decrease with increasing the PCR input volume, indicating that DNA purified from urine using Norgen's kit is free of the common inhibitors usually present in urine.

Figure 10. Purification of cell-free circulating RNA and exosomal RNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000) was used to purify cell-free circulating and exosomal RNA from 2 mL, 5 mL and 10 mL urine. 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 urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000) was used to purify RNA from 2 mL, 5 mL and 10 mL urine. 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 urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit was able to recover 95% of the 5S rRNA transcript and 93% of miR-21 from 5 mL urine relative to the amount that is present in 2 mL urine. Moreover, 92% of the 5S rRNA transcript and 93% of the miR-21 was recovered from 10 mL urine relative to the amount that is present in 5 mL urine.

Figure 12. Determination of the amount of inhibition present in urine RNA samples when detecting the human 5S transcript and miR-21. RNA was isolated from 2 mL, 5 mL and 10 mL urine using Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000). 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 urine using Norgen's kit is free of the common inhibitors usually present in urine.

Figure 13. Purification of cell-free circulating DNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100) was used to purify circulating DNA from 10 mL, 20 mL and 30 mL Urine. Two microliters of the purified NA was then used as the template in qPCR reactions to assess the relative amount of the purified 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 urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100) was used to purify circulating NA from 10 mL, 20 mL and 30 mL Urine. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the linearity of the purified housekeeping 5S rRNA gene from the different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit was able to recover 99% of the 5S rRNA gene from 20 mL urine relative to the amount that is present in 10 mL urine. Moreover, 98% of the 5S rRNA gene was recovered from 30 mL urine relative to the amount that is present in 20 mL urine.

Figure 15. Determination of the amount of inhibition present in urine cell-free circulating DNA samples when detecting the human 5S gene. DNA was isolated from 10 mL, 20 mL and 30 mL urine using Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100). 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. In fact, the Ct values tend to decrease with increasing the PCR input volume indicating that DNA purified from urine using Norgen's kit is free of the common inhibitors usually present in urine.

Figure 16. Purification of cell-free circulating RNA and exosomal RNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100) was used to purify cell-free circulating and exosomal RNA from 10 mL, 20 mL and 30 mL urine. 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 urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100) was used to purify RNA from 10 mL, 20 mL and 30 mL urine. 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 urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit was able to recover 94% of the 5S rRNA transcript and 97% of miR-21 from 20 mL urine relative to the amount that is present in 10 mL urine. Moreover, 98% of the 5S rRNA transcript and 94% of the miR-21 was recovered from 30 mL urine relative to the amount that is present in 20 mL urine.

Figure 18. Determination of the Amount of Inhibition Present in Urine RNA Samples when Detecting the Human 5S transcript and miR-21. RNA was isolated from 10 mL, 20 mL and 30 mL urine using Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100). 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 urine using Norgen's kit is free of the common inhibitors usually present in urine.

Kit Specifications
Minimum Urine Input	250 μL
Maximum Urine Input	2 mL
Size of Nucleic Acid Purified	All sizes, including miRNA and small RNA (< 200 nt)
Elution Volume	50-100 μL
Time to Complete 10 Purifications	25-30 minutes
Average Yields	Variable depending on specimen

Storage Conditions and Product Stability

All buffers should be kept tightly sealed and stored at room temperature. Theses kits are 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. 59900 (50 preps)	Cat. 60000 (20 preps)	Cat. 60100 (10 preps)
Binding Solution K	25 mL	75 mL	1 x 75 mL 1 x 25 mL
Lysis Buffer A	30 mL	20 mL	20 mL
Wash Solution A	18 mL	18 mL	18 mL
Elution Buffer F	6 mL	6 mL	6 mL
Mini Spin Columns	50	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