Plasma/Serum Exosome Purification and RNA Isolation Kits

Overview

Purification and enrichment of intact plasma/serum exosomes for functional studies
Isolate all sizes of RNA, including microRNA, irrespective of size or GC content, without bias
Versatile plasma/serum input volume
No phenol extractions, Proteinase K treatment, nor carrier RNA required
No time-consuming ultracentrifugation, filtration nor special syringes required
No precipitation reagents, nor overnight incubation required
Compatible with plasma/serum from most species
Pure exosomes are purified and are free from any other RNA-binding proteins
Purification is based on spin column chromatography that uses Norgen’s proprietary resin
The purified RNA is suitable for a variety of downstream applications, including Small RNA Sequencing. Find out more information on Norgen's NGS services

Norgen’s Plasma/Serum Exosome and RNA Isolation Kits constitute all-in-one systems for the purification of exosomes and the sequential isolation of exosomal RNA from different plasma/serum sample volumes. The purification is based on spin column chromatography that employs Norgen’s proprietary resin. These kits are designed to isolate all sizes of RNA, including microRNA, irrespective of size or GC content, without bias. These kits provide a clear advantage over other available kits in that they do not require any special instrumentation, protein precipitation reagents, extension tubes, phenol/chloroform or protease treatments. The RNA isolated from the purified exosomes is free from any protein-bound circulating RNA and is of the highest integrity. The purified RNA can be used in a number of downstream applications including real time PCR, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.

Norgen’s Plasma/Serum Exosome and RNA Isolation Mini Kit

For sample volumes ranging from 50 µL to 1 mL. This kit allows the user to elute into a flexible elution volume ranging from 50 µL to 100 µL.

Norgen’s Plasma/Serum Exosome and RNA Isolation Midi Kit

For sample volumes ranging from 1 mL to 4 mL. This kit allows the user to elute into a flexible elution volume ranging from 50 µL to 100 µL.

Norgen’s Plasma/Serum Exosome and RNA Isolation Maxi Kit

For sample volumes ranging from 4 mL to 10 mL. This kit allows the user to elute into a flexible elution volume ranging from 50 µL to 100 µL.

Details

Supporting Data

Figure 1. Isolation of RNA from exosomes purified from different plasma volumes. Norgen's Plasma/Serum Exosome Purification and RNA Isolation Mini Kit (Cat# 58300) was used to isolate RNA from exosomes purified from different plasma volumes using the same kit. 2 μL of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated plasma exosomal miR-26a. (A) The average Ct value for plasma exosomal miR-26a is linearly decreasing with increasing the sample input volume. (B) The relative amount of the plasma exosomal miR-26a shows excellent linearity with a percentage of recovery of more than 90%.

Figure 2. Comparing exosome yields from different purification methods. Intact exosomes were purified from 1 mL plasma using Norgen's Plasma/Serum Exosome Purification and RNA Isolation Mini Kit (Cat# 58300), Competitor S's kit and Competitor L's kit (from plasma). Exosomes purified using Norgen's kit were resuspended in 200 µL of Norgen's ExoR buffer, diluted 1:1,000 and visualized on the NanoSight LM10 instrument. The analysis shows that Norgen's kit isolated 55 nm exosomes with a recovery of 9.64 x 10¹³ particles/mL plasma. No impurities were found to be contaminating the exosomes purified using Norgen's Plasma/Serum Exosome Purification and RNA Isolation Kit. Additionally, exosomes with a broader size range covering from 50 nm - 150 nm were purified from 1 mL plasma with a higher concentration compared to the other two methods.

Figure 3. NanoSight data showing intact exosomes purified from 1 mL and 10 mL plasma. Intact exosomes were purified from 1 mL plasma using Norgen's Plasma/Serum Exosome Purification and RNA Isolation Mini Kit (Cat# 58300) and from 10 mL plasma using Norgen's Plasma/Serum Exosome Purification and RNA Isolation Maxi Kit (Cat# 58600). Exosomes purified using Norgen's mini kit were resuspended in 200 µL of Norgen's ExoR buffer whereas exosomes purified using Norgen's Maxi kit were resuspended in 600 µL Norgen's ExoR buffer, diluted 1:1,000 and visualized on the NanoSight LM10 instrument. The analysis shows that the purification of exosomes is linear as 4.04 x 10¹⁰ particles/mL were recovered from 1 mL plasma, whereas 2.95 x 10¹¹ particles/mL were recovered from 10 mL plasma.

Figure 4. Isolation of RNA from exosomes purified from different plasma volumes. Norgen’s Plasma/Serum Exosome Purification and RNA Isolation Midi Kit (Cat# 58500) was used to isolate RNA from exosomes purified from different plasma volumes using the same kit. 2 μL of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated plasma exosomal miR-26a. (A) The average Ct value for plasma exosomal miR-26a is linearly decreasing with increasing sample input volume. B) The relative amount of the plasma exosomal miR-26a shows excellent linearity with a percentage of recovery of more than 90%.

Figure 5. Isolation of RNA from exosomes purified from different plasma volumes. Norgen’s Plasma/Serum Exosome Purification and RNA Isolation Maxi Kit (Cat# 58600) was used to isolate RNA from exosomes purified from different plasma volumes using the same kit. 2 μL of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated plasma exosomal miR-26a. (A) The average Ct value for plasma exosomal miR-26a is linearly decreasing with increasing sample input volume. B) The relative amount of the plasma exosomal miR-26a shows excellent linearity with a percentage of recovery of more than 90%.

Kit Specifications
Minimum Plasma/Serum Input	50 μL
Maximum Plasma/Serum Input	1 mL
Size of Exosomes Purified	40 nm - 150 nm
Size of RNA Purified	All sizes, including miRNA and small RNA (< 200 nt)
Elution Volume	50-100 μL
Time to Complete 10 Purifications	35 - 40 minutes
Average Yields*	Variable depending on specimen

*Please check page 4 of the product insert for the average yields and the common RNA quantification methods.

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.

Important Note
This kit is suitable for the purification of exosomes 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.

Component	Cat. 58300 (50 preps)	Cat. 58500 (25 preps)	Cat. 58600 (15 preps)
Slurry E	12.5 mL	12.5 mL	12.5 mL
ExoC Buffer	8 mL	8 mL	2 x 8 mL
ExoR Buffer	12 mL	12 mL	12 mL
Lysis Buffer A	20 mL	20 mL	20 mL
Lysis Additive B	2 mL	2 mL	2 mL
Wash Solution A	38 mL	18 mL	18 mL
Elution Solution A	6 mL	6 mL	6 mL
Mini Filter Spin Columns	50	25	15
Mini Spin Columns	50	25	15
Collection Tubes	50	25	15
Elution Tubes (1.7 mL)	100	50	30
Product Insert	1	1	1

Documentation

RELATED BLOGS

FAQs

Mini, Midi, Maxi

What If a variable speed centrifuge is not available?

A fixed speed centrifuge can be used, however reduced yields may be observed.

At what temperature should I centrifuge my samples?

All centrifugation steps are performed at room temperature. Centrifugation at 4°C will not adversely affect kit performance.

What if I added more or less of the specified reagents’ volume?

Adding more or less than the specified volumes may reduce both the quality and the quantity of the purified RNA. Eluting your RNA in high volumes will increase the yield but will lower the concentration. Eluting in small volumes will increase the concentration but will lower the overall yield.

What if I forgot to do a dry spin before my final elution step?

Your purified RNA will be contaminated with the Wash Solution A. This may reduce the quality of your purified RNA and will interfere with your downstream applications.

Can I perform a second elution?

Yes, but it is recommended that the 2nd elution be in a smaller volume (50% of 1st Elution). It is also recommended to perform the 2nd elution into a separate elution tube to avoid diluting the 1st elution.

Why do my samples show low RNA yield?

Exosomes contain very little RNA. This varies from individual to individual based on numerous variables. In order to increase the yield, the amount of urine, plasma or serum input could be increased.

Why do the A260:280 ratio of the purified RNA is lower than 2.0?

Most of the Exosomal RNA is short RNA fragments with a very low concentration where the A260/280 ratio tends to decrease with the decrease in the RNA concentration. The A260:280 ratio is normally between 1-1.6. This low A260:280 ratio will not affect any downstream application.

Why does my isolated RNA not perform well in downstream applications?

If a different Elution Buffer was used other than the one provided in the kit, the buffer should be checked for any components that may interfere with the application. Common components that are known to interfere are high salts (including EDTA), detergents and other denaturants. Check the compatibility of your elution buffer with the intended use.

Citations

Mini (58300)

Title	Cancer cells exploit an orphan RNA to drive metastatic progression
Journal	Nature Medicine. 2018.
Authors	Lisa Fish, Steven Zhang, Johnny X. Yu, Bruce Culbertson, Alicia Y. Zhou, Andrei Goga & Hani Goodarzi

Maxi (58500)

Title	Small RNA-sequence analysis of plasma-derived extracellular vesicle miRNAs in smokers and patients with chronic obstructive pulmonary disease as circulating biomarkers
Journal	Journal of Extracellular Vesicles, 8(1). 2019.
Authors	Sundar, I. K., Li, D., & Rahman, I.

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Plasma/Serum Exosome Purification and RNA Isolation Kits