Figure 2. Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit was used to purify circulating DNA from 200 µL, 300 µL and 400 µL plasma prepared from blood collected on citrate as an anticoagulant. 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 plasma volumes. Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit was able to recover 98% of the 5S rRNA gene from 300 µL plasma relative to the amount that is present in 200 µL plasma. Moreover, 98% of the 5S rRNA gene was recovered from 400 µL plasma relative to the amount that is present in 300 µL plasma.

Figure 3.  DNA was isolated from 200 µL, 300 µL and 400 µL plasma using Norgen's Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit. 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. Norgen's Plasma/Serum cell-free circulating DNA Purification Maxi Kit was used to purify circulating DNA from 5 mL, 8 mL and 10 mL plasma prepared from blood collected on citrate as an anticoagulant in comparison to Competitor Q's kits. Two milliliters 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 increases linearly with increasing the sample input volume. Norgen's kit showed the most consistent and the highest recovery of the housekeeping 5S rRNA gene as compared to the other isolation method.

Figure 5.  Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit was used to purify circulating DNA from 5 mL, 8 mL and 10 mL plasma prepared from blood collected on citrate as an anticoagulant. Two milliliters 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 plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit was able to recover 94% of the 5S rRNA gene from 8 mL plasma relative to the amount that is present in 5 mL plasma. Moreover, 96% of the 5S rRNA gene was recovered from 10 mL plasma relative to the amount that is present in 10 mL plasma.

Figure 6.  DNA was isolated from 5 mL, 8 mL and 10 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit. 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 7.  Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit was used to purify circulating DNA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant, and compared to Competitor Q's kit. 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 increases linearly with increasing the sample input volume. Norgen's kit showed the most consistent and the highest recovery of the housekeeping 5S rRNA gene as compared to the other isolation method.

Figure 8.  Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit as used to purify circulating DNA from 50 µL, 100 µL and 200 µL plasma prepared from blood collected on citrate as an anticoagulant. 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 plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit was able to recover 96% of the 5S rRNA gene from 100 µL plasma relative to the amount that is present in 50 µL plasma. Moreover, 97% of the 5S rRNA gene was recovered from 200 µL plasma relative to the amount that is present in 100 µL plasma.

Figure 9.  DNA was isolated from 50 µL, 100 µL and 200 µL plasma using Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit. 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 DNA from different plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit  was used to purify circulating DNA from 0.5 mL, 1 mL, 2 mL and 4 mL plasma prepared from blood collected on citrate as an anticoagulant in comparison to Competitor Q's kits. 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 increases linearly with increasing the sample input volume. Norgen's Kit showed the most consistent and the highest recovery of the housekeeping 5S rRNA gene as compared to the other isolation method.

Figure 11. Linearity of DNA purified from increasing plasma volumes using Norgen’s Plasma/Serum cell-free circulating DNA Purification Midi Kit.  Norgen’s Plasma/Serum cell-free circulating DNA Purification Midi Kit was used to purify circulating DNA from 0.5 mL, 1 mL, 2 mL and 4 mL plasma prepared from blood collected on citrate as an anticoagulant. 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 plasma volumes. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit was able to recover 97% of the 5S rRNA gene from 1 mL plasma relative to the amount that is present in 0.5 mL plasma. Moreover, 95% of the 5S rRNA gene was recovered from 2 mL plasma relative to the amount that is present in 1 mL plasma. Additionally, 95% of the 5S rRNA gene was recovered from 4 mL plasma relative to the amount that is present in 2 mL plasma.

Figure 12. Determination of the amount of inhibition present in plasma cell-free circulating DNA samples when detecting the human 5S gene.  DNA was isolated from 0.5 mL, 1 mL, 2 mL and 4 mL plasma using Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit. 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.