Plasma and serum RNA purification kit

Plasma/Serum RNA Purification Kits provide a fast and simple method for isolating circulating RNA and exosomal RNA from plasma and serum samples.

Format

Mini

50 Preps

Image Credit: Norgen Biotek Corporation

Midi

20 Preps

Image Credit: Norgen Biotek Corporation

Maxi

10 Preps

Image Credit: Norgen Biotek Corporation

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Features and benefits

• Separate circulating and exosomal RNA of all sizes, including microRNA
• Variable ranges of plasma/serum inputs
• No phenol extractions
• Absence of carrier RNA
• Bind and elute all RNA without bias, regardless of size or GC content
• Concentrate exosomal and circulating RNA into a flexible elution volume
• Compatible with Streck Cell-Free RNA BCT^® Tubes
• Purified, high-quality RNA can be used for small RNA sequencing and other downstream processes.
• Purification is based on spin column chromatography, which uses Norgen's exclusive resin separation matrix.

These kits offer a quick, dependable, and practical way to use a handy spin column method to purify and concentrate high-quality, high-purity, inhibitor-free, cell-free circulating and exosomal RNA. Fresh or frozen serum or plasma samples made from blood drawn on either EDTA or citrate can have their RNA purified using these kits.

It is not recommended to use plasma samples made from blood obtained while on heparin because heparin can seriously disrupt several subsequent processes, including RT-PCR.

All downstream procedures, such as PCR, qPCR, methylation-sensitive reverse transcription qPCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, expression array assays, and NGS, are completely compatible with the purified plasma/serum RNA.

Background

Exosomal RNA, or plasma/serum cell-free circulating RNA, may offer biomarkers for specific cancers and disease conditions. Most cell types secrete exosomes, membrane vesicles ranging in size from 40 to 150 nm.

Exosomes are present in saliva, blood, urine, amniotic fluid, and malignant ascitic fluids, among other biological fluids. Recently, there has been mounting evidence that these vesicles serve as cellular messengers, transmitting information to distant bodily tissues and cells.

These exosomes might be involved in tissue repair, neural communication, pathogenic protein transfer, and adaptive immune responses to infections and tumors. Exosomal RNAs may, therefore, be used as biomarkers for several illnesses, including cancer. Since RNAses prevent the RNA molecules enclosed in exosomes from degrading, they can be effectively extracted from biological fluids like serum or plasma.

Plasma/serum RNA purification mini kit

With volumes ranging from 50 µL to 200 µL, this kit can purify RNA from fresh or frozen serum or plasma samples made from blood obtained on either EDTA or citrate. The final volume, ranging from 10 µL to 25 µL, is where the purified plasma/serum RNA is eluted.

Plasma/serum RNA purification midi kit

Using a two-column technique, RNA can be separated from frozen or fresh serum or plasma samples from blood drawn on citrate or EDTA in volumes between 250 µL and 1.5 mL. For a final elution of 50 µL to 100 µL, the first column will handle the large volume input of bodily fluids, which will then be concentrated on a mini-column.

Plasma/serum RNA purification maxi kit

RNA can be isolated using this kit from fresh or frozen serum or plasma from blood obtained using either citrate or EDTA. Plasma samples made from blood obtained while on heparin are not recommended because heparin can seriously disrupt a number of subsequent processes, including RT-PCR.

Image Credit: Norgen Biotek Corporation

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Details

Supporting data

Figure 1. Purification of circulating RNA from different plasma volumes. Norgen's Plasma/Serum RNA Purification Mini Kit was used to purify circulating RNA from 50 µL, 100 µL, and 200 µL plasma prepared from blood collected on EDTA. Three microlitres of the purified RNA were then used as the template in RT-qPCR reactions to detect miR-21 (Figure 1A) and the housekeeping 5S rRNA transcript (Figure 1B). The relative amount of both the miR-21 (Figure 1A) and the 5S rRNA transcript (Figure 1B) is linearly increasing with increasing the sample input volume. Image Credit: Norgen Biotek Corporation

Figure 2. Eluting purified circulating RNA into different elution volumes. Norgen’s 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. Image Credit: Norgen Biotek Corporation

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. Image Credit: Norgen Biotek Corporation

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 with Plasma/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. Image Credit: Norgen Biotek Corporation

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. Image Credit: Norgen Biotek Corporation

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. Image Credit: Norgen Biotek Corporation

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 Norgen’s Total RNA Purification Kit recovers a higher number of miRNAs than 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 detected to compare Norgen and the competitor’s recovery of miRNA. Norgen’s 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. Image Credit: Norgen Biotek Corporation

Figure 8. Purification of cell-free circulating RNA and exosomal RNA from different plasma volumes. Norgen’s 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. Image Credit: Norgen Biotek Corporation

Figure 9. Linearity of RNA purified from increasing plasma volumes using Norgen’s Plasma/Serum RNA Purification Midi Kit. Norgen’s 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. Norgen’s 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. Image Credit: Norgen Biotek Corporation

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 Norgen’s 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 Norgen’s kit is free of the common inhibitors usually present in plasma. Image Credit: Norgen Biotek Corporation

Figure 11. Purification of cell-free circulating RNA and exosomal RNA from different plasma volumes. Norgen’s 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. Image Credit: Norgen Biotek Corporation

Figure 12. Linearity of RNA purified from increasing plasma volumes using Norgen’s Plasma/Serum RNA Purification Maxi Kit. Norgen’s 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. Norgen’s 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, Norgen’s 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. Image Credit: Norgen Biotek Corporation

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 Norgen’s 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 Norgen’s kit is free of the common inhibitors usually present in plasma. Image Credit: Norgen Biotek Corporation

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Mini

Source: Norgen Biotek Corporation

*This kit is suitable for the isolation of RNA from fresh or frozen serum or plasma prepared from blood collected on either EDTA or Citrate. Plasma samples prepared from blood collected on heparin should not be used as heparin can significantly interfere with many downstream applications such as RT-PCR.
**Please check page 7 for Average Plasma/Serum Yields and Common RNA Quantification Methods.

Midi

Source: Norgen Biotek Corporation

*Please check page 7 of the Product Insert for Average Plasma/Serum Yields and Common RNA Quantification Methods.

Maxi

Source: Norgen Biotek Corporation

*Please check page 7 of the Product Insert for Average Plasma/Serum Yields and Common RNA Quantification Methods.

This kit is designed for RNA isolation from fresh or frozen serum or plasma prepared from blood collected using EDTA or Citrate. Plasma samples from blood collected with heparin should be avoided, as heparin can interfere with downstream applications such as RT-PCR.

Storage conditions and product stability

Every solution needs to be stored at room temperature and kept tightly sealed. For two years following the date of shipment, this kit remains stable. If any salt precipitation is seen, Lysis Buffer A should be warmed for 20 minutes at 60 °C.

Source: Norgen Biotek Corporation