Urine DNA Isolation Kits

Features and Benefits

Rapid isolation of both small and large species of DNA from urine
Convenient spin column format
Effective removal of PCR inhibitors
Purified DNA is highly suited to sensitive downstream applications
Allows for the purification of viral DNA from urine

Both high molecular weight DNA (greater than 1 kb in size; mostly cell associated) and the smaller DNA (150 - 250 bp; derived from the circulation) is effectively isolated and purified using a rapid and convenient spin column protocol. This kit can be used to isolate DNA from a broad range of viruses in urine as well. Salts, metabolic wastes, proteins and other contaminants are removed to yield inhibitor-free DNA for use in sensitive applications. The DNA is of excellent quality for various downstream applications such as PCR, qPCR and DNA fingerprinting, methylation studies and more.

This kit is fully compatible with Norgen's Urine Collection and Preservation Tubes.

Urine DNA Isolation Kit

This kit provides a fast, reliable and simple procedure for isolating DNA from urine volumes ranging from 50 μL to 1.75 mL of urine. Preparation time for a single sample is about 30 minutes.

Urine DNA Isolation Kit (Slurry Format)

This kit provides a fast, reliable and simple procedure for isolating DNA from urine volumes ranging from 3 mL to 25 mL. Preparation time for a single sample is less than 30 minutes.

Urine DNA Isolation Maxi Kit (Slurry Format)

This kit provides a fast, reliable and simple procedure for isolating DNA from urine volumes ranging from 25 mL of urine up to 80 mL. Preparation time for a single sample is less than 30 minutes.

Background

DNA found in urine can be divided into 2 basic categories. The larger species, genomic-DNA (gDNA), is generally greater than 1 kb in size, and appears to be derived mainly from exfoliated cells. The second species is smaller, generally between 150 and 250 bp (apoptotic-DNA), and derives, at least in part, from the circulation. The second species is also considered as an RNA/DNA hybrid as reported by Halicka et al. (2000). Both types of DNA can be isolated reliably using this kit.

Details

Supporting Data

Figure 1. A Typical Agarose Gel Showing Total Urinary DNA Isolated from 1.5 mL of Urine using Norgen's Urine DNA Isolation Kit. Total urinary DNA was isolated from two different 1.5 mL urine samples. The samples were processed according to the bind, wash and elute procedure provided in the kit, and the DNA was eluted into 2 separate elution volumes. The proteins were first eluted into 100 µL of Elution Buffer (E1), followed by a second elution using 75 µL of Elution Buffer (E2). The purified urine DNA was then run on a 1.2% agarose gel, and each ane contains one tenth from each elution (i.e. E1: 10 µL out of 100 µL were loaded on the gel, E2: 7.5 µL out of 75 µL were loaded on the gel ). Lane M is 10 µL of Norgen’s FastRunner DNA Ladder. Both the large and small urine DNA species can be seen on the gel.

Figure 2. Isolation and Detection of DNA from 1.75 mL Urine Samples. Total genomic DNA was isolated from two different 1.75 mL urine samples using Norgen’s Urine DNA Isolation Kit. The bind, wash and elute procedure was performed, and the purified DNA was eluted into two separate elutions of 100 µL (E1) and 75 µL (E2). The isolated DNA was then subjected to quantitative PCR using human 5S gene primers to detect the genomic DNA using the iQ SYBR Green Supermix (BioRad, #170-8882). Five microliters from each elution was used as a template in a 20 µL qPCR reaction. The red line in the PCR baseline graph above corresponds to the first elution from the DNA isolated from the first urine sample, the green line corresponds to the second elution from the DNA isolated from first urine sample, the blue line corresponds to the first elution from the DNA isolated from the second urine sample, whereas the orange line corresponds to the second elution from the DNA isolated from the second urine sample. The yellow line corresponds to the No Template Control.

Figure 3. Circulating DNA Isolated from Urine can be used as the Template in PCR Reactions.Total urinary DNA was isolated from three different 1.5 mL urine samples using Norgen's Urine DNA Isolation Kit. The bind, wash and elute procedure was performed, and the purified DNA was eluted into two separate elutions of 100 µL (E1) and 75 µL (E2). Five microliters of each elution was then used as a template in a PCR reaction to amplify the K-ras gene. Lanes A-C contain the expected 157 bp product, and correspond to the first elution from each sample. Lane D is the positive control of 293 HEK DNA and shows the expected 157 bp product, while Lane E is the negative control. Lane M is Norgen's FastRunner DNA Ladder.

Figure 4. Typical Agarose Gel Showing Total Urinary DNA Isolated from Different Urine Volumes using Norgen's Urine DNA Isolation Kit (Slurry Format). Total urinary DNA was isolated from 3 mL, 6 mL, 12 mL and 24 mL of urine. Total urinary DNA was isolated from each urine sample according to the isolation protocol that is optimized for different sample volumes. The isolated DNA was eluted into two separate elutions (E1 and E2). The purified urine DNA was then loaded onto a 1.5% agarose gel. Each lane shows one tenth from each elution. It can be seen that the first elution contains most of urinary DNA whereas the second elution contains the rest of the urinary DNA isolated. Lane M is 10 µL of Norgen's FastRunner DNA Ladder.

Figure 5. Isolation and Detection of DNA from Different Volumes of Urine. Total genomic DNA was isolated from 3, 6, 12 and 24 mL urine samples using Norgen's Urine DNA Isolation Kit (Slurry Format). The urinary DNA was isolated from each urine sample according to the provided protocol, and the DNA was eluted into two separate elutions (E1 and E2). The isolated DNA was then subjected to quantitative PCR using human 5S gene primers to detect the genomic DNA. To test the absence of PCR inhibitors usually accompanying DNA isolated from urine, an increasing amount from each elution (1, 3, 6 and 9 µL) was used as a template in the PCR reaction. Urinary genomic DNA was successfully detected from all the different urine volumes with no sign of PCR inhibition, even when 9 µL of urine DNA was used as the template.

Figure 6. Highly Sensitive Isolation of Viral DNA from 25 mL of Urine. Herpes Simplex Virus - 1 (HSV-1) sequences were cloned into a plasmid, and 25 mL urine samples were then spiked with decreasing amounts of the plasmid (10⁶, 10⁵, 10⁴, 10³, 10² and 10¹). Total DNA was then isolated using Norgen’s Urine DNA Isolation Kit (Slurry Format). Ten microlitres of each 100 µL elution was then used as the template in a 20 µL PCR reaction using HSV-1-specific primers and Norgen's 2X PCR Master Mix (Cat #28007). Each PCR reaction was then run on a 1.5% agarose gel for visual analysis. The HSV-1 amplification product is 275 bp. As few as 250 HSV-1 viral copies could be isolated and detected from 25 mL of urine.

Figure 7. Typical Agarose Gel Showing Total Urinary DNA Isolated from Different Urine Volumes using Norgen's Urine DNA Isolation Maxi Kit (Slurry Format). Total urinary DNA was isolated from 20 mL, 40 mL, 60 mL and 80 mL of urine. Total urinary DNA was isolated from each urine sample according to the isolation protocol that is optimized for different sample volumes. The isolated DNA was eluted, and was then loaded onto a 1.8% agarose gel. Each lane shows one tenth from each elution. It can be seen that urine DNA is increasing linearly with the urine input volume. It should also be noted that the circulating DNA started to appear in the form of ladder with the increase of the urine sample input. Lane M is 10 µL of Norgen's FastRunner DNA Ladder.

Figure 8. Linearity of Ct Values and DNA Yields When Scaling Up Urine Input. Total urinary DNA was isolated from 20 mL, 40 mL, 60 mL and 80 mL of urine. Total urinary DNA was isolated from each urine sample according to the isolation protocol that is optimized for different sample volumes. The isolated DNA was then subjected to quantitative PCR using human 5S gene primers to detect the genomic DNA. Urinary genomic DNA was successfully detected from all the different urine volumes with no sign of PCR inhibition and in a linear relationship to the total urine DNA yield (Ct values decrease with increasing the sample urine input)

Kit Specifications
Volume of Urine Processed	1.75 mL
Average Yield*	Up to 50 ng
Size of DNA Purified	Large (> 1 kb) and small (150-250 bp)
Time to Complete 10 Purifications	30 minutes hands-on time (plus a 1 hour incubation)

* Yield will vary depending on the type of sample processed

Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature. This kit is 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. 18100 (50 preps)	Cat. 48800 (50 preps)	Cat. 50100 (50 preps)
Binding Solution K	15 mL	-	-
Slurry B1	-	18 mL	18 mL
Proteinase K in Storage Buffer	2 mL	-	-
Pronase K in Storage Buffer	2 mL	-	-
Soluton WN	9 mL	-	-
Binding Buffer A	-	-	50 mL
Lysis Buffer A	-	30 mL	30 mL
Wash Solution A	-	38 mL	38 mL
Wash Solution B	30 mL	-	-
Wash Solution D	9 mL	-	-
Binding Solution K	15 mL	-	-
Elution Buffer B	15 mL	15 mL	15 mL
Micro Spin Columns	50	-	-
Mini Filter Spin Columns	-	50	50
Collection Tubes	50	50	50
Elution Tubes (1.7 mL)	100	100	100
Product Insert	1	1	1

Documentation

FAQs

Spin Column, Slurry, Maxi Slurry

If I am not going to process my samples immediately, how should I store my samples?

We recommend the use of Norgen’s Urine Preservative when collecting urine samples, which is designed for the preservation of nucleic acids and proteins in fresh urine samples at ambient temperatures. Urine samples in the preservative should be stored at room temperature. Turbidity or precipitation may be observed if the urine samples are stored at either 4°C or at -20°C. DO NOT discard this precipitate and/or spin down your samples to get rid of the turbidity; this will significantly reduce your DNA yields. Make sure to mix your samples thoroughly before processing.

What if a variable speed centrifuge is not available?

A fixed speed centrifuge can be used, however reduced yields may be observed.

What will happen if my centrifugation speed varied from the recommended speed?

This may lead to the degradation of the genomic DNA or reduction in the total DNA yields.

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.

My centrifuge speeds are defined in rpm and not in g-force. How can I convert g-force to rpm?

The correct rpm can be calculated using the formula:

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.

Can I process a different urine volume?

Yes, you can. This protocol is outlined for an input of 1.75 mL of urine, however it is possible to process 50 μL – 2.6 mL of urine. Ensure that the volume of Binding Solution K added is adjusted accordingly based on the urine input.

What if I added more or less from the specified reagents’ volume?

Adding less volume may reduce your DNA yields. Adding more may not affect the DNA yields EXCEPT if more Elution Buffer B was added. Eluting DNA in a higher volume of Elution Buffer B will result in diluting your DNA.

What if my incubation varied from the 20 minutes specified in the product manual?

Less than 20 minutes will result in a lower DNA yields. More than 20 minutes may not affect your DNA yields.

What if I forgot to do a dry spin after my second wash?

Your first DNA elution will be contaminated with traces of the Wash Solutions. This may dilute the DNA yield in your first elution. Also, it may interfere with your downstream applications. Re-isolate the eluted DNA using the same procedure as you initially isolated the DNA from urine but using the first elution as your input.

Can I perform a third elution?

Yes, you can. A third elution is possible, but it is recommended that this elution be performed in a smaller volume (50 μL).

Why am I eluting my DNA into two elutions using the 18100 kit?

The first elution will mainly contain the low nucleic acid species and traces from the large DNA species. However, your second elution will mainly contain the large DNA species and traces from the low nucleic acid species.

Why did my samples show very low DNA yields?

Some urine samples contain very little DNA. This varies from individual to individual based on numerous variables. In order to increase the yield, the amount of urine input could be increased. Increasing the incubation time at 65°C (up to overnight) could also result in increased yields.

Why does my DNA not perform well in downstream applications?

If a different Elution Buffer B 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 B with the intended use.

Can I use frozen Urine samples for isolating DNA?

Yes, you can use Norgen's Urine DNA isolation kit for forzen Urine samples. However, urine samples stored at -80°C, -20°C or at 4°C can develop some precipitation due to the aggregation of some of the highly abundant proteins in urine. Eliminating these precipitates using centrifugation or filtration may cause the loss of some of the protein-bound cf-NA or exosomes. Furthermore, these precipitates may affect the quality of the purified nucleic acids.

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 - 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.

I am noticing a white precipitate in my elution. What should I do?

This white precipitate may appear in the elution depending on the nature of the sample. This white precipitate will not affect your downstream application. Simply mix your elution well before using.

Citations

Title	Performance of the NG OligoGen kit for the diagnosis of Neisseria gonorrhoeae: comparison with cobas 4800
Citation	Diagnostic Microbiology and Infectious Disease 2015.
Authors	M. Parra-Sánchezcorrespondenceemail, S. García-Rey1email, A. Marcuello2email, I. Zakariya-Yousef1email, S. Bernal1email, I. Pueyo3email, E. Martín-Mazuelos4email, J.C. Palomares

Title	A noninvasive assay for monitoring renal allograft status
Citation	Journal of Biological Methods 2014.
Authors	V Romanov, TC Whyard, WC Waltzer, FS Darras

Title	Long-Term Frozen Storage of Urine Samples: A Trouble to Get PCR Results in Schistosoma spp. DNA Detection?
Citation	PLoS One 2013.
Authors	P. Fermandez-Soto, V. Velasco Tirado, C. Carranza Rodriguez, J.L. Perez-Arellano, A. Muro

Title	RIG-I detects infection with live Listeria by sensing secretd bacterial nucleic acids
Citation	The EMBO Journal 2012.
Authors	Abdullah, Z., Schlee, M., Roth, S., Mraheil, M. A., Barchet, W., Böttcher, J., ... & Civril, F

Title	Methods for Separate Isolation of Cell-Free DNA and Cellular DNA from Urine-Application of Methylation-Specific PCR on both DNA Fractions
Citation	The Open Biomarkers Journal 2011.
Authors	Agnes Beermann, Foued Ghanjati, Thomas Hermanns, Cedric Poyet, Joana Pereira, Johannes Fischer, Peter Wernet, Simeon Santourlidis

Title	Renal kallikrein excretion and epigenetics in human acute kidney injury: Expression, mechanisms and consequences
Citation	BMC Nephrology 2011.
Authors	Sun Woo Kanga,, Pei-an Betty Shihb,, Roy O. Mathewc,*, Manjula Mahatab, Nilima Biswasb, Fangwen Raob, Liying Yand, Josee Boucharde, Rakesh Malhotraf, Ashita Tolwanig, Srikrishna Khandrikaf, Ravindra L. Mehtaf,†, Daniel T. O'Connorb,f,†.