Figure 2. Linearity of DNA purified from increasing urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 59900) was used to purify circulating NA from 500 µL, 1 mL and 2 mL urine. Two microlitres of the purified NA 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 and Viral Nucleic Acid Purification Mini Kit was able to recover 91% of the 5S rRNA gene from 1 mL urine relative to the amount that is present in 500 µL urine. Moreover, 95% 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 and Viral Nucleic Acid Purification Mini Kit (Cat# 59900). 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 RNA and exosomal RNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 59900) was used to purify cell-free circulating and exosomal RNA from 500 µL, 1 mL and 2 mL urine. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of the purified (A) 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 and Viral Nucleic Acid Purification Mini Kit (Cat# 59900) was used to purify RNA from 500 µL, 1 mL and 2 mL urine. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the linearity of the purified (A) housekeeping 5S rRNA transcript and (B) miR-21 from the different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit was able to recover 92% of the 5S rRNA transcript and 95% of miR-21 from 1 mL urine relative to the amount that is present in 500 µL urine. Moreover, 92% of the 5S rRNA transcript and 92% of the miR-21 was recovered from 2 mL urine relative to the amount that is present in 1 mL urine. 

Figure 6. Determination of the amount of inhibition present in urine RNA samples when detecting the human 5S transcript and miR-21. RNA was isolated from 500 µL, 1 mL and 2 mL urine using Norgen’s Urine Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 59900). Increasing volumes of the elution (2, 4 and 8 μL) were used in a 20 μL reverse transcription reaction followed by qPCR amplification 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 the PCR input volume used as a template in the reverse transcription reaction did not affect the Ct value generated from the qPCR amplification for both the (A) 5S rRNA transcript and (B) miR-21. 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 DNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000) was used to purify circulating DNA from 2 mL, 5 mL and 10 mL Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the relative amount of the purified housekeeping 5S rRNA gene. The Ct values for the 5S rRNA gene are linearly decreasing with increasing the sample input volume.

Figure 8. Linearity of DNA purified from increasing urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000) was used to purify circulating NA from 2 mL, 5 mL and 10 mL urine. Two microlitres of the purified NA 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 and Viral Nucleic Acid Purification Midi Kit was able to recover 92% of the 5S rRNA gene from 5 mL urine relative to the amount that is present in 2 mL urine. Moreover, 99% of the 5S rRNA gene was recovered from 10 mL urine relative to the amount that is present in 5 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 2 mL, 5 mL and 10 mL urine using Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000). 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 10. Purification of cell-free circulating RNA and exosomal RNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000) was used to purify cell-free circulating and exosomal RNA from 2 mL, 5 mL and 10 mL urine. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of the purified (A) 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 11. Linearity of RNA purified from increasing urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000) was used to purify RNA from 2 mL, 5 mL and 10 mL urine. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the linearity of the purified (A) housekeeping 5S rRNA transcript and (B) miR-21 from the different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit was able to recover 95% of the 5S rRNA transcript and 93% of miR-21 from 5 mL urine relative to the amount that is present in 2 mL urine. Moreover, 92% of the 5S rRNA transcript and 93% of the miR-21 was recovered from 10 mL urine relative to the amount that is present in 5 mL urine.

Figure 12. Determination of the amount of inhibition present in urine RNA samples when detecting the human 5S transcript and miR-21. RNA was isolated from 2 mL, 5 mL and 10 mL urine using Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 60000). Increasing volumes of the elution (2, 4 and 8 µL) were used in a 20 µL reverse transcription reaction followed by qPCR amplification 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 the PCR input volume used as a template in the reverse transcription reaction did not affect the Ct value generated from the qPCR amplification for both (A) 5S rRNA transcript and (B) miR-21. 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 13. Purification of cell-free circulating DNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100) was used to purify circulating DNA from 10 mL, 20 mL and 30 mL Urine. Two microliters of the purified NA was then used as the template in qPCR reactions to assess the relative amount of the purified housekeeping 5S rRNA gene. The Ct values for the 5S rRNA gene is linearly decreasing with increasing the sample input volume.

Figure 14. Linearity of DNA purified from increasing urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100) was used to purify circulating NA from 10 mL, 20 mL and 30 mL Urine. Two microlitres of the purified NA 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 and Viral Nucleic Acid Purification Maxi Kit was able to recover 99% of the 5S rRNA gene from 20 mL urine relative to the amount that is present in 10 mL urine. Moreover, 98% of the 5S rRNA gene was recovered from 30 mL urine relative to the amount that is present in 20 mL urine.

Figure 15. 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 and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100). 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 16. Purification of cell-free circulating RNA and exosomal RNA from different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100) was used to purify cell-free circulating and exosomal RNA from 10 mL, 20 mL and 30 mL urine. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of the purified (A) 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 17. Linearity of RNA purified from increasing urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100) was used to purify RNA from 10 mL, 20 mL and 30 mL urine. Two microlitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the linearity of the purified (A) housekeeping 5S rRNA transcript and (B) miR-21 from the different urine volumes. Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit was able to recover 94% of the 5S rRNA transcript and 97% of miR-21 from 20 mL urine relative to the amount that is present in 10 mL urine. Moreover, 98% of the 5S rRNA transcript and 94% of the miR-21 was recovered from 30 mL urine relative to the amount that is present in 20 mL urine.

Figure 18. Determination of the Amount of Inhibition Present in Urine RNA Samples when Detecting the Human 5S transcript and miR-21. RNA was isolated from 10 mL, 20 mL and 30 mL urine using Norgen's Urine Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 60100). Increasing volumes of the elution (2, 4 and 8 μL) were used in a 20 μL reverse transcription reaction followed by qPCR amplification 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 the PCR input volume used as a template in the reverse transcription reaction did not affect the Ct value generated from the qPCR amplification for both (A) 5S rRNA transcript and (B) miR-21. 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.