Urine Cell-Free Circulating DNA Purification Kits

Supporting Data

Figure 1. Purification of cell-free circulating DNA from different urine volumes. Norgen’s Urine Cell-Free Circulating DNA Purification Mini Kit (Cat# 56600) was used to purify circulating DNA from 500 µL, 1 mL and 2 mL fresh urine. 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 is linearly increasing 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. Linearity of DNA purified from increasing urine volumes. Norgen’s Urine Cell-Free Circulating DNA Purification Mini Kit (Cat# 56600) was used to purify circulating DNA from 500 µL, 1 mL and 2 mL fresh urine. 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 urine volumes. Norgen’s Urine Cell-Free Circulating DNA Purification Mini Kit was able to recover 97% of the 5S rRNA gene from 1 mL urine relative to the amount that is present in 500 µL Urine. Moreover, 92% 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 DNA Purification Mini Kit (Cat# 56600). 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 DNA from different urine volumes. Norgen's Urine Cell-Free Circulating DNA Purification Midi Kit (Cat# 55700) was used to purify circulating DNA from 2 mL, 5 mL and 10 mL fresh urine. 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 is linearly increasing with increasing the sample input volume. Norgen's kit showed the most consistent and the highest recovery of the housekeeping 5S rRNA gene.

Figure 5. Linearity of DNA purified from increasing urine volume. Norgen's Urine Cell-Free Circulating DNA Purification Midi Kit (Cat# 55700) was used to purify circulating DNA from 2 mL, 5 mL and 10 mL fresh urine. 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 urine volumes. Norgen's Urine Cell-Free Circulating DNA Purification Midi Kit was able to recover 99% of the 5S rRNA gene from 5 mL urine relative to the amount that is present in 1 mL Urine. Moreover, 93% of the 5S rRNA gene 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 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 DNA Purification Midi Kit (Cat# 55700). 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 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 DNA Purification Maxi Kit (Cat# 56800) was used to purify circulating DNA from 10 mL, 20 mL and 30 mL fresh urine. 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 is linearly increasing with increasing the sample input volume. Norgen's kit showed the most consistent and the highest recovery of the housekeeping 5S rRNA gene.

Figure 8. Linearity of DNA purified from increasing urine volumes. Norgen's Urine Cell-Free Circulating DNA Purification Maxi Kit (Cat# 56800) was used to purify circulating DNA from 10 mL, 20 mL and 30 mL fresh urine. Two microlitres of the purified DNA was then used as the template in qPCR reactions to assess the linearity of the purified the housekeeping 5S rRNA gene from the different urine volumes. Norgen's Urine Cell-Free Circulating DNA Purification Maxi Kit was able to recover 100% of the 5S rRNA gene from 20 mL urine relative to the amount that is present in 10 mL urine. Moreover, 100% of the 5S rRNA gene 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 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 DNA Purification Maxi Kit (Cat# 56800). 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.

Kit Specifications
Minimum Urine Input	250 μL
Maximum Urine Input	2 mL
Size of DNA Purified	All sizes of DNA ≥ 50 bp
Elution Volume	50-100 μL
Time to Complete 10 Purifications	15-20 minutes
Average Yields	Variable depending on specimen

*Please check page 6 of the product insert for 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. These kits are 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. 56600 (50 preps)	Cat. 56700 (20 preps)	Cat. 56800 (10 preps)
Binding Solution K	25 mL	75 mL	1 x 75 mL 1 x 25 mL
Proteinase K	1.2 mL	0.3 mL	0.3 mL
Wash Solution A	38 mL	2 x 38 mL	38 mL
Elution Buffer B	8 mL	15 mL	15 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

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FAQs

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°C 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 temperature varied from the specified 55°C?

The incubation temperature can be in the range of 55°C - 60°C. If the temperature is outside of that range the activity of the Proteinase K will be reduced. This will result in a reduction in your DNA yields.

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?

Urine samples contain very little cfc-DNA. This varies from individual to individual. In order to increase the yield, the amount of urine 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.

Do I need to do an RNase treatment for my DNA Elution?

Norgen's Urine Cell-Free Circulating DNA doesn't co-purify urine circulating RNA along with circulating DNA, therefore an RNase step is not required.

Why are the A260:280 ratio and the A260:230 ratio of the purified DNA low?

Most of the urine DNA is present in short fragments as well as in a very low concentration. The low A260:280 ratio and the low A260:230 ratio will not affect any downstream applications.

Is spinning down the sample necessary before beginning cfDNA/cfRNA isolation from urine?

Yes, preparing the cell-free urine sample is a very important step as it removes host and bacterial cells and debris that can contaminate the purification and downstream processes.

I lost / finished the proteinase K in storage buffer from my Norgen Biotek kit. Can I purchase it separately?

Yes, you can purchase more Proteinase K and it is supplied in a pack size of 5 x 1mL tubes. Please contact us and request for Product number 28229.

Can I use frozen Urine samples for isolating cfc-DNA?

For cfc-DNA isolation, the samples should have been clarified to prepare cell-free samples before freezing them.

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.

Urine Cell-Free Circulating DNA Purification Kits