Urine Cell-Free Circulating RNA Purification Kits
For research use only and NOT intended for in vitro diagnostics.
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Urine Cell-Free Circulating RNA Purification Kits
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Supporting Data
Figure 1. Purification of cell-free circulating RNA and exosomal RNA from different urine volumes. Norgen's Urine Cell-Free Circulating RNA Purification Mini Kit (Cat# 56900) was used to purify cell-free circulating and exosomal RNA from 0.5 mL, 1 mL and 2 mL urine samples. Two microlilitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of (A) the 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 2. Linearity of RNA purified from increasing urine volumes. Norgen's Urine Cell-Free Circulating RNA Purification Mini Kit (Cat# 56900) was used to purify RNA from 0.5 mL, 1 mL and 2 mL urine samples. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the linearity of the (A) 5S rRNA transcript and (B) miR-21 from the different urine volumes. Norgen's Urine Cell-Free Circulating RNA Purification Mini Kit was able to recover 96% of the 5S rRNA transcript from 1 mL urine relative to the amount that is present in 0.5 mL plasma. Moreover, 96% of the 5S rRNA transcript was recovered from 2 mL urine relative to the amount that is present in 1 mL urine. As for miR-21, Norgen's Urine Cell-Free Circulating RNA Purification Mini Kit was able to recover 92% of miR-21 from 1 mL urine relative to the amount that is present in 0.5 mL urine. Furthermore, 100% of miR-21 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 RNA samples when detecting the human miR-21. RNA was isolated from 0.5 mL, 1 mL and 2 mL urine using Norgen's Urine Cell-Free Circulating RNA Purification Mini Kit (Cat# 56900). 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 RNA 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 RNA Purification Midi Kit (Cat# 57000) was used to purify cell-free circulating and exosomal RNA from 2 mL, 5 mL and 10 mL urine samples. Two microlilitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of (A) the 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 RNA Purification Midi Kit (Cat# 57000) was used to purify RNA from 2 mL, 5 mL and 10 mL urine samples. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the linearity of the (A) 5S rRNA transcript and (B) miR-21 from the different urine volumes. Norgen's Urine Cell-Free Circulating RNA Purification Midi Kit was able to recover 96% of the 5S rRNA transcript from 5 mL urine relative to the amount that is present in 2 mL plasma. Moreover, 97% of the 5S rRNA transcript was recovered from 10 mL urine relative to the amount that is present in 5 mL urine. As for miR-21, Norgen's Urine Cell-Free Circulating RNA Purification Midi Kit was able to recover 98% of miR-21 from 5 mL urine relative to the amount that is present in 1 mL urine. Furthermore, 92% of miR-21 was recovered from 10 mL urine relative to the amount that is present in 5 mL urine.
Figure 6. Determination of the amount of inhibition present in urine cell-free circulating RNA samples when detecting the human miR-21. RNA was isolated from 2 mL, 5 mL and 10 mL urine using Norgen's Urine Cell-Free Circulating RNA Purification Midi Kit (Cat# 57000). 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 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 RNA and exosomal RNA from different urine volumes. Norgen’s Urine Cell-Free Circulating RNA Purification Maxi Kit (Cat# 57100) was used to purify cell-free circulating and exosomal RNA from 10 mL, 20 mL and 30 mL urine samples. Two microlilitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of (A) the 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 8. Linearity of RNA purified from increasing urine. Norgen’s Urine Cell-Free Circulating RNA Purification Maxi Kit (Cat# 57100) was used to purify RNA from 10 mL, 20 mL and 30 mL urine samples. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the linearity of the(A) 5S rRNA transcript and (B) miR-21 from the different urine volumes. Norgen’s Urine Cell-Free Circulating RNA Purification Maxi Kit was able to recover 95% of the 5S rRNA transcript from 20 mL urine relative to the amount that is present in 10 mL Urine. Moreover, 99% of the 5S rRNA transcript was recovered from 30 mL urine relative to the amount that is present in 20 mL urine. As for miR-21, Norgen’s Urine Cell-Free Circulating RNA Purification Maxi Kit was able to recover 99% of miR-21 from 20 mL urine relative to the amount that is present in 10 mL urine. Furthermore, 96% of miR-21 was recovered from 30 mL urine relative to the amount that is present in 20 mL urine.
Figure 9. Determination of the amount of inhibition present in urine cell-free circulating RNA samples when detecting the human miR-21. RNA was isolated from 10 mL, 20 mL and 30 mL urine using Norgen’s Urine Cell-Free Circulating RNA Purification Maxi Kit (Cat# 57100). 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 RNA purified from urine using Norgen’s kit is free of the common inhibitors usually present in urine.
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Kit Specifications
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| Sample Volume Range |
250 μL to 2 mL
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| Size of RNA Purified |
All sizes including small RNA (< 200 nt)
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| Minimum Elution Volume |
50 μL
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| Maximum Elution Volume |
100 μL
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| Time to Complete 10 Purifications |
25-30 minutes
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| Average Yield* |
Variable depending on specimen
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*Please check page 6 of the product insert for average yields and the common RNA quantification methods.
Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature. These 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. 56900 (50 preps) | Cat. 57000 (20 preps) | Cat. 57100 (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 Solution A | 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 |
Documentation
FAQs
Mini, Midi, Maxi
RPM= √RCF/(1.118x10-5)(r)
Where RCF = required gravitational acceleration (relative centrifugal force in units of g); r = radius of the rotor in cm; and RPM = the number of revolutions per minute required to achieve the necessary g-force.
We recommend the following steps to prepare frozen urine for isolation:
- Gently warm the sample to room temperature or 37°C for 5 min.
- DO NOT perform a centrifugation step after thawing frozen cell-free Urine samples - this will eliminate the precipitated proteins leading to loss of protein-bound cf-NA or exosomes.
- Proceed with the protocol.
We recommend the use of Norgen’s Urine Preservative when collecting urine samples. Norgen’s Urine Preservative is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures, therefore no protein precipitation will occur and the purified nucleic acids will be of a higher quality.
