High Efficiency Real-Time PCR from Urine DNA
Posted: Thu 21 Jul 2016 09:26High Efficiency Real-Time PCR from Urine DNA
NORGEN - Biotech Corporation
Application Note 34 - Urine DNA Preservation
INTRODUCTION
In recent years attention has been turning to the use of non-invasive samples for genetic and diagnostic analysis, including the use of urine. Norgen Biotek Corp. has developed a simple method for the concentration, preservation, shipping, storage and isolation of DNA from urine using Individual Urine DNA Concentration and Preservation Devices. Donors simply collect their urine and transfer it directly into the Urine Concentration Tube, and then mix the urine with Norgen’s resin that is inside the tube. All the macromolecules present within the urine will bind to the resin in the presence of the buffer located within the Urine Concentration Tube. Next, the resin is then allowed to precipitate by gravity for 10 minutes, then the urine supernatant is discarded and Norgen’s Urine Preservative is added. The preservative is an aqueous storage buffer designed for rapid preservation of urine DNA from fresh, concentrated urine specimens.
This buffer stabilizes the DNA for long-term storage at ambient temperature. Since the buffer prevents the growth of microorganisms and inactivates viruses it also allows the samples to be handled and shipped safely. The DNA subsequently isolated from the preserved samples is of a high quality and can be used directly in sensitive downstream diagnostic assays such as real-time PCR. Real-time PCR is one major application that is used in quantification of gene copy numbers, viral quantitation, pathogen detection as well as array verification and genotyping. In this study DNA was collected and isolated from 20 donors using Norgen’s Urine DNA Concentration, Preservation and Isolation kit, and the DNA was used as the template in real-time PCR reactions to show the high quality of the isolated DNA
MATERIALS AND METHODS
DNA Isolation
Twenty different urine samples were collected using Norgen’s Urine DNA Concentration, Preservation and Isolation Kit. DNA was purified from 30 mL of the concentrated urine/preservative mix according to the supplied protocol.
Real-Time PCR Amplification
Two microliter of each DNA urine sample was then used as the template in a real-time PCR reaction using 2XSYBR Green/Fluorescein qPCR Master Mix. 5S specific primers were used in the reaction amplified on the iCycler iQ realtime system (Bio-Rad).
RESULTS AND DISCUSSION
Urine DNA must be pure and free from inhibitors in order for the DNA to be used in sensitive downstream applications. Here, DNA was collected and isolated from 20 urine samples using Norgen’s Urine DNA Concentration, Preservation and Isolation kit, and was subsequently used as the template in Real-Time PCR reactions to detect the 5S gene. Figure 1 shows both the successful and consistent amplification of all 20 DNA samples (Panel A), and the melt curve indicates real amplification from all templates (Panel B). Figure 2 shows a graph of the Ct values from all the DNA samples, and the consistent values indicate that the DNA samples were all of a similar quality. Therefore, all 20 DNA samples were amplifiable on real-time PCR and the average Ct value was 18+0.41.
Figure 1. Amplification of the 5S Gene from 20 Urine DNA samples. (A) Real-time PCR amplification and (B) melt-curve analysis. DNA samples are in Red color and the no template control is colored in Blue. All the DNA templates were successfully amplified with high efficiency.
Figure 2: Ct values of the real-time amplification of 5S gene from the 20 Urine DNA samples. The average Ct value is 18+0.41.
CONCLUSION
DNA isolated using Norgen’s Urine DNA Concentration, Preservation and Isolation Kit is of a very high quality, and is compatible with PCR applications, including real-time PCR. All 20 saliva DNA samples were amplified with similar Ct values, indicating the consistency of the DNA isolation.
NORGEN - Biotech Corporation
Application Note 34 - Urine DNA Preservation
INTRODUCTION
In recent years attention has been turning to the use of non-invasive samples for genetic and diagnostic analysis, including the use of urine. Norgen Biotek Corp. has developed a simple method for the concentration, preservation, shipping, storage and isolation of DNA from urine using Individual Urine DNA Concentration and Preservation Devices. Donors simply collect their urine and transfer it directly into the Urine Concentration Tube, and then mix the urine with Norgen’s resin that is inside the tube. All the macromolecules present within the urine will bind to the resin in the presence of the buffer located within the Urine Concentration Tube. Next, the resin is then allowed to precipitate by gravity for 10 minutes, then the urine supernatant is discarded and Norgen’s Urine Preservative is added. The preservative is an aqueous storage buffer designed for rapid preservation of urine DNA from fresh, concentrated urine specimens.
This buffer stabilizes the DNA for long-term storage at ambient temperature. Since the buffer prevents the growth of microorganisms and inactivates viruses it also allows the samples to be handled and shipped safely. The DNA subsequently isolated from the preserved samples is of a high quality and can be used directly in sensitive downstream diagnostic assays such as real-time PCR. Real-time PCR is one major application that is used in quantification of gene copy numbers, viral quantitation, pathogen detection as well as array verification and genotyping. In this study DNA was collected and isolated from 20 donors using Norgen’s Urine DNA Concentration, Preservation and Isolation kit, and the DNA was used as the template in real-time PCR reactions to show the high quality of the isolated DNA
MATERIALS AND METHODS
DNA Isolation
Twenty different urine samples were collected using Norgen’s Urine DNA Concentration, Preservation and Isolation Kit. DNA was purified from 30 mL of the concentrated urine/preservative mix according to the supplied protocol.
Real-Time PCR Amplification
Two microliter of each DNA urine sample was then used as the template in a real-time PCR reaction using 2XSYBR Green/Fluorescein qPCR Master Mix. 5S specific primers were used in the reaction amplified on the iCycler iQ realtime system (Bio-Rad).
RESULTS AND DISCUSSION
Urine DNA must be pure and free from inhibitors in order for the DNA to be used in sensitive downstream applications. Here, DNA was collected and isolated from 20 urine samples using Norgen’s Urine DNA Concentration, Preservation and Isolation kit, and was subsequently used as the template in Real-Time PCR reactions to detect the 5S gene. Figure 1 shows both the successful and consistent amplification of all 20 DNA samples (Panel A), and the melt curve indicates real amplification from all templates (Panel B). Figure 2 shows a graph of the Ct values from all the DNA samples, and the consistent values indicate that the DNA samples were all of a similar quality. Therefore, all 20 DNA samples were amplifiable on real-time PCR and the average Ct value was 18+0.41.
Figure 1. Amplification of the 5S Gene from 20 Urine DNA samples. (A) Real-time PCR amplification and (B) melt-curve analysis. DNA samples are in Red color and the no template control is colored in Blue. All the DNA templates were successfully amplified with high efficiency.
Figure 2: Ct values of the real-time amplification of 5S gene from the 20 Urine DNA samples. The average Ct value is 18+0.41.
CONCLUSION
DNA isolated using Norgen’s Urine DNA Concentration, Preservation and Isolation Kit is of a very high quality, and is compatible with PCR applications, including real-time PCR. All 20 saliva DNA samples were amplified with similar Ct values, indicating the consistency of the DNA isolation.