Plasma/Serum Exosome and Free-Circulating RNA Isolation Kits

Features and Benefits

Isolate all sizes of RNA, including microRNA, irrespective of size or GC content, without bias.
Versatile sample input ranges
Isolate all sizes of free-circulating RNA, including microRNA
The purified exosomal RNA is free from any circulating RNA-binding proteins
No phenol extractions, Proteinase K treatment, nor carrier RNA
No time-consuming ultracentrifugation, filtration nor special syringes are required
No precipitation reagents, nor overnight incubation required
Concentrate isolated exosomal RNA and are free-circulating RNA into a flexible elution volume ranging from 50 µL to 100 µL
Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

These kits provide a fast, reliable and convenient method to sequentially isolate and concentrate exosomal RNA as well as Free-Circulating 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 as well as all sizes of the free-circulating protein-bound RNA, including microRNA. 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. Moreover, these kits allow the user to elute into a flexible elution volume ranging from 50 µL to 100 µL. The RNA isolated from the purified exosomes is free from any protein-bound circulating RNA and is of the highest integrity. Moreover, the free-circulating, protein-bound, RNA is free from any exosomal RNA. 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.

Plasma/Serum Exosome and Free-Circulating RNA Isolation Mini Kit

For sample volumes ranging from 50 µL to 1 mL.

Plasma/Serum Exosome and Free-Circulating RNA Isolation Midi Kit

For sample volumes ranging from 1 mL to 4 mL.

Plasma/Serum Exosome and Free-Circulating RNA Isolation Maxi Kit

For sample volumes ranging from 4 mL to 10 mL.

Details

Supporting Data

Figure 1. Isolation of RNA from exosomes purified from different plasma volumes. Norgen’s Plasma/Serum Exosome and Free-Circulating RNA Isolation Mini Kit (Cat# 59500) was used to isolate Exosomal RNA from different plasma volumes ranging from 0.1 mL and up to 1 mL. Two microlitres 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 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. Isolation of Free-Circulating RNA after the isolation of Exosomal RNA from different plasma volumes. Norgen’s Plasma/Serum Exosome and Free-Circulating RNA Isolation Mini Kit (Cat# 59500) was used to isolate Free-Circulating RNA after the isolation of Exosomal RNA from different plasma volumes ranging from 0.1 mL and up to 1 mL. Two microlitres of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated plasma free-circulating miR-21. (A) The free-circulating miR-21 is linearly decreasing with increasing the sample input volume. B) The relative amount of the free-circulating miR-21 shows excellent linearity with a percentage of recovery of more than 90%.

Figure 3. Isolation of RNA from exosomes purified from different plasma volumes. Norgen’s Plasma/Serum Exosome and Free-Circulating RNA Isolation Midi Kit (Cat# 59600) was used to isolate Exosomal RNA from different plasma volumes ranging from 1 mL and up to 4 mL. Two microlitres 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 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 4. Isolation of Free-Circulating RNA after the isolation of Exosomal RNA from different plasma volumes. Norgen’s Plasma/Serum Exosome and Free-Circulating RNA Isolation Midi Kit (Cat# 59600) was used to isolate Free-Circulating RNA after the isolation of Exosomal RNA from different plasma volumes ranging from 1 mL and up to 4 mL. Two microlitres of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated plasma free-circulating miR-21. (A) The free-circulating miR-21 is linearly decreasing with increasing the sample input volume. B) The relative amount of the free-circulating miR-21 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 and Free-Circulating RNA Isolation Maxi Kit (Cat# 59700) was used to isolate exosomal RNA from different plasma volumes ranging from 4 mL and up to 10 mL. Two microlitres 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 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 6. Isolation of Free-Circulating RNA after the isolation of Exosomal RNA from different plasma volumes. Norgen’s Plasma/Serum Exosome and Free-Circulating RNA Isolation Maxi Kit (Cat# 59700) was used to isolate free-circulating RNA after the isolation of exosomal RNA from different plasma volumes ranging from 4 mL and up to 10 mL. Two microlitres of the isolated RNA was then used as the template in RT-qPCR reactions to assess the amplification of the isolated plasma free-circulating miR-21. (A) The free-circulating miR-21 is linearly decreasing with increasing the sample input volume. B) The relative amount of the free-circulating miR-21 shows excellent linearity with a percentage of recovery of more than 90%.

Kit Specifications
Minimum Plasma Input	50 μL
Maximum Plasma Input	1 mL
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 5 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. 59500 (50 preps)	Cat. 59600 (25 preps)	Cat. 59700 (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	2 x 20 mL	2 x 20 mL	2 x 20 mL
Lysis Additive B	2 mL	2 mL	2 mL
Wash Solution A	2 x 18 mL	18 mL	18 mL
Elution Solution A	2 x 6 mL	6 mL	6 mL
Mini Filter Spin Columns	50	25	15
Mini Spin Columns	100	50	30
Collection Tubes	100	50	30
Elution Tubes (1.7 mL)	100	50	30
Product Insert	1	1	1

Documentation

PROTOCOLS

(59500) Plasma/Serum Exosome and Free-Circulating RNA Isolation Mini Kit - Protocol (50 prep)

(59600) Plasma/Serum Exosome and Free-Circulating RNA Isolation Midi Kit - Protocol (25 prep)

(59700) Plasma/Serum Exosome and Free-Circulating RNA Isolation Maxi Kit - Protocol (15 prep)

SAFETY DATA SHEETS

59500 - Plasma/Serum Exosome and Free Circulating RNA Isolation Mini Kit - SDS

59600 - Plasma/Serum Exosome and Free Circulating RNA Isolation Midi Kit - SDS

59700 - Plasma/Serum Exosome and Free Circulating RNA Isolation Maxi Kit - SDS

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

Title	Comparative Analysis of Free-Circulating and Vesicle-Associated Plasma microRNAs of Healthy Controls and Early-Stage Lung Cancer Patients
Journal	Pharmaceutics. 2022.
Authors	Luigi Pasini, Ivan Vannini, Paola Ulivi, Michela Tebaldi, Elisabetta Petracci, Francesco Fabbri, Franco Stella and Milena Urbini

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Plasma/Serum Exosome and Free-Circulating RNA Isolation Kits