Exosome Purification & RNA Isolation Tutorial - Plasma/Serum

In this tutorial, you will learn how to use Norgen Biotek’s Plasma/Serum Exosome Purification and RNA Isolation Mini Kit (Cat. 58300) for the purification and enrichment of intact plasma/serum exosomes for functional studies.
https://norgenbiotek.com/product/plas...

Before you begin, sterilize your workstation and all instruments using 70% ethanol followed by 1% bleach. After this, you are ready to begin the procedure.

In this kit, you will find:
A detailed product insert, 1.7 mL Elution Tubes, Mini Spin Columns, Mini-Filter Spin Columns, Wash Solution A, Slurry E, Lysis Buffer A, ExoR Buffer, Elution Solution A, ExoC Buffer, and Lysis Additive B.

Please refer to the “notes prior to use” section in the protocol before beginning. https://norgenbiotek.com/product/plas...

Preparation of Cell-Free Plasma/Serum from Frozen Sample
1. Place your frozen Plasma/Serum at 4oC to thaw.
2. After thawing your plasma/serum sample, aliquot the volume to be processed and centrifuge at 2 minutes at 400 x g (~2,000 RPM).
3. After centrifugation, transfer the clear plasma/serum supernatant to a fresh tube. Cell-Free Plasma/Serum is now ready for Exosomes purification.

Section 1: Exosome Purification from 50 µL - 1 mL Cell-Free Plasma/Serum
1. To 1 mL plasma/serum add 3 mL Nuclease-free water followed by the addition of 100 µL of ExoC Buffer. (Note: The final volume of any plasma/serum sample to be processed should be 4 mL before the addition of the specified 100 µL of ExoC Buffer)
2. To the mixture from Step 1 add 200 µL of Slurry E. Mix well by vortexing for 10 seconds and let stand at room temperature for 5 minutes. (Note: Mix Slurry E well prior to use. For optimal performance ensure that resin is completely resuspended).
3. Mix well by vortexing for 10 seconds. Centrifuge for 2 minutes at 2,000 RPM. Discard the supernatant.
4. Apply 200 µL ExoR Buffer to the slurry pellet and mix well by vortexing for 10 seconds.
5. Incubate the slurry pellet resuspended in the 200 µL ExoR Buffer at room temperature for 5 minutes.
6. After incubation, mix well by vortexing for 10 seconds then centrifuge for 2 minutes at 500 RPM.
7. Transfer the supernatant to a Mini Filter Spin column assembled in an elution tube and centrifuge for 1 minute at 6,000 RPM. Do Not Discard the flowthrough which contains your purified Exosomes. Your Exosomes are now ready for RNA Isolation (Section 2) or any other downstream applications.

Section 2: Exosomal RNA Isolation
1. Add 300 µL of Lysis Buffer A and 37.5 µL of Lysis Additive B to the 200 µL ExoR Buffer containing the purified Exosomes (Section 1, Step 7).
2. Mix well by vortexing for 10 seconds then incubate at room temperature for 10 minutes
3. After incubation add 500 µL of 96-100% Ethanol to the mixture from Step 2 and mix well by vortexing for 10 seconds.
4. Transfer 500 µL of the mixture from Step 3 into a Mini Spin Column. Centrifuge for 1 minute at 3,300 x g (~6,000 RPM). Discard the flowthrough and reassemble the spin column with its collection tube.
5. Repeat Step 4 one more time to transfer the remaining mixture from Step 3 into the Mini Spin Column
6. Apply 600 µL of Wash Solution A to the column and centrifuge for 30 seconds at 3,300 x g (~6,000 RPM). Discard the flowthrough and reassemble the spin column with its collection tube.
7. Repeat Step 6 one more time, for a total of two washes.
8. Spin the column, empty, for 1 minute at 13,000 x g (~14,000 RPM). Discard the collection tube.
9. Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 50 µL of Elution Solution A to the column and centrifuge for 1 minute at 2,000 RPM, followed by 2 minutes at 8,000 RPM.
10. For maximum recovery, transfer the eluted buffer back to the column and let stand at room temperature for 2 minutes. Centrifuge for 1 minute at 400 x g (~2,000 RPM), followed by 2 minutes at 5,800 x g (~8,000 RPM).

Exosomal RNA is now ready for downstream applications!

To learn more about this kit, please visit our website https://norgenbiotek.com/product/plas...

Or, give us a call at 1-866-NORGENB (Toll-free) or email our product specialists at [email protected].

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