Figure 2. Eluting purified circulating RNA into different elution volumes. Norgens Plasma/Serum RNA Purification Mini Kit was used to purify circulating RNA from 200 µL plasma prepared from blood collected on EDTA and eluted in 10 µL, 15 µL, and 25 µL. Three microlitres of the purified RNA was then used as the template in RT-qPCR reactions to detect miR-21 (Figure 2A) and the housekeeping 5S rRNA transcript (Figure 2B). The relative amount of both the miR-21 (Figure 2A) and the 5S rRNA transcript (Figure 2B) is increasing with increasing the elution volume indicating the efficient concentration of the plasma circulating RNA in a very low elution volume.

Figure 3. Effective and consistent detection of miRNA from plasma. Norgen's Plasma/Serum RNA Purification Mini Kit can effectively isolate miRNA from plasma. Circulating miRNA was isolated from 200 µL plasma using Norgen's Plasma/Serum RNA Purification Mini Kit, competitor Q's kit and competitor E's kit. Circulating miRNA was isolated from 600 µL using competitor A's kit. Stem loop RT-qPCR using primers specific to miR-21 was performed. In brief, 1 microliter of the 15 µL RNA purified using Norgen's Plasma/Serum RNA Purification Mini Kit, competitor Q's kit and 3.3 microliters of the 50 µL purified RNA using competitor E's kit and competitor A's kit was then subjected to a 20 µL reverse transcription using miR-21 stem-loop reverse primer. Three microliters of the reverse transcription was used in a 20 µL real-time PCR reaction with primers to detect the human miR-21. Norgen's Plasma/Serum RNA Purification Mini Kit showed the most consistent and the highest recovery of the miR-21 transcripts as compared to the other isolation methods. The recovery of the miRNA from 200 µL plasma using Norgen's kit was higher than that recovered from RNA purified from 600 µL using competitor A's kit.

Figure 4. Small RNA Sequencing from as little as 50 µL of Plasma.Norgen Biotek has developed an effective pipeline for small RNA sequencing from small volumes of plasma/serum. RNA can be effectively and consistently recovered from as little as 50 µL of plasma using Norgen's patented sample preparation technology (example shown here withPlasma/Serum RNA Purification Mini Kit, Cat. 55000). Panel A shows that the number of microRNA detected from 50 or 200 µL of plasma was almost identical to that of 4 mL. Panel B is a Venn diagram showing that of the microRNAs identified, the majority are detected in all volumes of plasma input from 50 µL to 4 mL. In fact, the scatter plots in Panel C show that the relative expression level of each microRNA detected was highly correlated between 50 or 200 µL of plasma and 4 mL plasma.

Figure 5. High Degree of Overlapping of miRNA profile between Urine and Plasma. Plasma and mid-stream urine was collected from three different healthy individuals. RNA was isolated from 20 mL of each urine sample using Norgen's Urine Exosomal RNA Purification Kit (Cat. 47200) and 200 µL of each plasma sample using Norgen's Plasma/Serum RNA Purification Mini Kit (Cat. 55000). Small RNA Libraries were then generated using an Illumina TruSeq Small RNA Library Preparation Kit and subsequently sequenced on an Illumina MiSeq system. The list of mapped miRNAs from plasma and urine from the same individual was then compared. The Venn diagrams showed that the miRNA profiles have high degree of overlapping between urine and plasma within each individual.

Figure 6. Increased Diversity of Other Small RNA Species (Piwi-Interacting) in Urine. Plasma and mid-stream urine was collected from three different healthy individuals. RNA was isolated from 20 mL of each urine sample using Norgen's Urine Exosomal RNA Purification Kit (Cat. 47200) and 200 µL of each plasma sample using Norgen's Plasma/Serum RNA Purification Mini Kit (Cat. 55000). Small RNA Libraries were then generated using an Illumina TruSeq Small RNA Library Preparation Kit and subsequently sequenced on an Illumina MiSeq system. The above chart showed that the relative proportion of piwi-interacting RNA (piRNA) was consistently higher in urine.

Figure 7. Better Diversity of miRNA Detected from HeLa Cells using Illumina Small RNA Next Gen Sequencing.Norgen's Total RNA Purification Kit isolates miRNA from HeLa cells with better diversity than a leading competitor. Total RNA including miRNA was isolated from 1 million HeLa cells using Norgen's Total RNA Purification Kit and Competitor Q's leading miRNA Kit, and was applied to Illumina Small RNA Next Gen Sequencing on a MiSeq sequencer. Panel A showed that Norgens Total RNA Purification Kit recovers ahigher number of miRNAsthan the competitor. In particular, the higher diversity is achieved with a faster and simpler procedure in as little as 20 minutes of RNA sample preparation time without the use of phenol. Panel B is a scatter plot of the average RPM (reads per million) of the miRNAs detectedto compare Norgen and the competitorsrecovery of miRNA. Norgens Total RNA Purification Kit recovered a significantly higher number of miRNAs that have higher RPM, as summarized in the graph insert as well as in Panel C.

Figure 8. Purification of cell-free circulating RNA and exosomal RNA from different plasma volumes. Norgens Plasma/Serum RNA Purification Midi Kit (Cat# 56100) was used to purify cell-free circulating and exosomal RNA from 0.25 mL, 0.75 mL 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 amplification of the (A) housekeeping 5S rRNA transcript and (B) miR-21. The average Ct value for both the 5S rRNA transcript and the miR-21 is linearly decreasing with increasing the sample input volume.

Figure 9. Linearity of RNA purified from increasing plasma volumes using Norgens Plasma/Serum RNA Purification Midi Kit. Norgens Plasma/Serum RNA Purification Midi Kit (Cat# 56100) was used to purify RNA from 0.25 mL, 0.7 mL 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 (A) the housekeeping 5S rRNA transcript and (B) miR-21 from the different plasma volumes. Norgens Plasma/Serum RNA Purification Midi Kit was able to recover 97% of both the 5S rRNA transcript and the miR-21 transcript from 0.75 mL plasma relative to the amount that is present in 0.35 mL plasma. Moreover, 95% of the 5S rRNA transcript and the miR-21 was recovered from 1.5 mL plasma relative to the amount that is present in 0.75 mL plasma.

Figure 10. Determination of the amount of inhibition present in plasma RNA samples when detecting the human 5S transcript and miR-21. RNA was isolated from 0.25 mL, 0.75 mL and 1.5 mL plasma using Norgens Plasma/Serum RNA Purification Midi Kit (Cat# 56100). 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 Norgens kit is free of the common inhibitors usually present in plasma.

Figure 11. Purification of cell-free circulating RNA and exosomal RNA from different plasma volumes. Norgens Plasma/Serum RNA Purification Maxi Kit (Cat# 56200) was used to purify cell-free circulating and exosomal RNA from 1.5 mL, 3 mL and 5 mL plasma prepared from blood collected on citrate as an anticoagulant. Two millilitres of the purified RNA was then used as the template in RT-qPCR reactions to assess the amplification of (A) the housekeeping 5S rRNA transcript and (B) miR-21. The average Ct value for both the 5S rRNA transcript and the miR-21 is linearly decreasing with increasing the sample input volume.

Figure 12. Linearity of RNA purified from increasing plasma volumes using Norgens Plasma/Serum RNA Purification Maxi Kit. Norgens Plasma/Serum Cell-Free Circulating and Exosomal RNA Purification Maxi Kit (Cat# 56200) was used to purify RNA from 1.5 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 (A) the housekeeping 5S rRNA transcript and (B) miR-21 from the different plasma volumes. Norgens Plasma/Serum RNA Purification Maxi Kit was able to recover 92% of the 5S rRNA transcript from 3 mL plasma relative to the amount that is present in 1.5 mL plasma. Moreover, 90% of the 5S rRNA transcript was recovered from 5 mL plasma relative to the amount that is present in 3 mL plasma. As for miR-21, Norgens Plasma/Serum RNA Purification Maxi Kit was able to recover 91% of miR-21 from 3 mL plasma relative to the amount that is present in 1.5 mL plasma. Furthermore, 90% of miR-21 was recovered from 5 mL plasma relative to the amount that is present in 3 mL plasma.

Figure 13. Determination of the amount of inhibition present in plasma RNA samples when detecting the human 5S transcript and miR-21. DNA was isolated from 1.5 mL, 3 mL and 5 mL plasma using Norgens Plasma/Serum RNA Purification Maxi Kit (Cat# 56200). 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 Norgens kit is free of the common inhibitors usually present in plasma.