Figure 2. Linearity of DNA purified from increasing plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300) was used to purify circulating NA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the linearity of the purified the housekeeping 5S rRNA gene from the different plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit was able to recover ninety-four percent of the 5S rRNA gene from 100 µL plasma relative to the amount that is present in 50 µL plasma. Moreover, 98% of the 5S rRNA gene was recovered from 200 µL plasma relative to the amount that is present in 100 µL plasma.

Figure 3. Determination of the amount of inhibition present in plasma cell-free circulating DNA samples when detecting the human 5S gene. DNA was isolated from 50 µL, 100 µL and 200 µL plasma using Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300). 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 plasma using Norgen's kit is free of the common inhibitors usually present in plasma.

Figure 4. Purification of cell-free circulating RNA and exosomal RNA from different plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300) was used to purify cell-free circulating and exosomal RNA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant in comparison. 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 plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300) was used to purify RNA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant. 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 plasma volumes. Norgen's Plasma/Serum Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit was able to recover 91% of the 5S rRNA transcript and 95% of miR-21 from 100 µL plasma relative to the amount that is present in 50 µL plasma. Moreover, 90% of the 5S rRNA transcript and 92% of the miR-21 was recovered from 200 µL plasma relative to the amount that is present in 100 µL plasma.

Figure 6. Determination of the amount of inhibition present in plasma RNA samples when detecting the human 5S transcript and miR-21. RNA was isolated from 50 µL, 100 µL and 200 µL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Mini Kit (Cat# 56300). 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 plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.

Figure 7. Purification of cell-free circulating DNA from different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400) was used to purify circulating DNA from 250 µL, 750 µL and 1.5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the relative amount of the purified the 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 plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400) was used to purify circulating NA from 250 µL, 750 µL and 1.5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the linearity of the purified the housekeeping 5S rRNA gene from the different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit was able to recover 98% of the 5S rRNA gene from 750 µL plasma relative to the amount that is present in 250 µL plasma. Moreover, 93% of the 5S rRNA gene was recovered from 1.5 mL plasma relative to the amount that is present in 750 µL plasma.

Figure 9. Determination of the amount of inhibition present in plasma cell-free circulating DNA samples when detecting the human 5S gene. DNA was isolated from 250 µL, 750 µL and 1.5 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400). 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 plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.

Figure 10. Purification of cell-free circulating RNA and exosomal RNA from different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400) was used to purify cell-free circulating and exosomal RNA from 250 µL, 750 µL and 1.5 mL plasma prepared from blood collected on citrate as an anticoagulant in comparison. 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 plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400) was used to purify RNA from 250 µL, 750 µL and 1.5 mL plasma prepared from blood collected on citrate as an anticoagulant. 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 plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit was able to recover 97% of both the 5S rRNA transcript and miR-21 from 750 µL plasma relative to the amount that is present in 250 µL plasma. Moreover, ninety-four percent of the 5S rRNA transcript and 97% of the miR-21 was recovered from 1.5 mL plasma relative to the amount that is present in 750 µL plasma.

Figure 12. Determination of the amount of inhibition present in plasma RNA samples when detecting the human 5S transcript and mir-21. RNA was isolated from 250 µL, 750 µL and 1.5 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Midi Kit (Cat# 56400). 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 plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.

Figure 13. Purification of cell-free circulating DNA from different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500) was used to purify circulating DNA from 2 mL, 3 mL and 5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the relative amount of the purified the 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 plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500) was used to purify circulating NA from 2 mL, 3 mL and 5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two microlitres of the purified NA was then used as the template in qPCR reactions to assess the linearity of the purified the housekeeping 5S rRNA gene from the different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit was able to recover 98% of the 5S rRNA gene from 3 mL plasma relative to the amount that is present in 2 mL plasma. Moreover, 93% of the 5S rRNA gene was recovered from 5 mL plasma relative to the amount that is present in 3 mL plasma.

Figure 15. Determination of the amount of inhibition present in plasma cell-free circulating DNA samples when detecting the human 5S gene. DNA was isolated from 2 mL, 3 mL and 5 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500). 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 plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.

Figure 16. Purification of cell-free circulating RNA and exosomal RNA from different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500) was used to purify cell-free circulating and exosomal RNA from 2 mL, 3 mL and 5 mL plasma prepared from blood collected on citrate as an anticoagulant in comparison. 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 plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500) was used to purify RNA from 2 mL, 3 mL and 5 mL plasma prepared from blood collected on citrate as an anticoagulant. 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 plasma volumes. Norgen’s Plasma/Serum Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit was able to recover 97% of both the 5S rRNA transcript and miR-21 from 3 mL plasma relative to the amount that is present in 2 mL plasma. Moreover, 94% of the 5S rRNA transcript and 97% of the miR-21 was recovered from 5 mL plasma relative to the amount that is present in 3 mL plasma.

Figure 18. Determination of the amount of inhibition present in plasma RNA samples when detecting the human 5S transcript and mir-21. RNA was isolated from 2 mL, 3 mL and 5 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating and Viral Nucleic Acid Purification Maxi Kit (Cat# 56500). 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 plasma using Norgen’s kit is free of the common inhibitors usually present in plasma.