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.