Authors: Irene Yan & Tushar Patel
Abstract
Extracellular vesicles can be isolated from different types of body fluids such as serum. These extracellular vesicles may contain protein, mRNA, and non-coding RNA. Extracellular vesicle RNA isolated from serum could be useful as a biomarker of disease. This protocol presents sample collection, process, and extracellular RNA isolation of serum using three different commercially available kits.
Reagents
- One of the three commercial kits:
- miCURY RNA Isolation Kit –Biofluids (Exiqon, #300112)
- miRNeasy Mini Kit (Qiagen, #217004)
- SeraMir Exosome RNA Purification Kit (System Biosciences, #RA806TC-1)
- Isopropanol
- Absolute ethanol
- Nuclease free H2O
Equipment
- Serum Tube (BD, 367988)
- 15 ml centrifuge tubes
- Bench top centrifuge
- Vortex Mixer
- Micropipettes with sterile tips 20-1000
- Sterile collection bottle
- Microcentrifuge tubes
Procedure
- Collect 10ml blood in serum collection tube.
- Allow blood to clot at room temperature for 1 hour.
- Centrifuge tube at 3000 rpm for 10s min at room temperature.
- Aliquot 1 ml serum into microcentrifuge tubes being careful to avoid disturbing the interface.
- Store samples at -70°C until ready for use.
- RNA Isolation can be performed from one of the following options.
A. miCURY RNA Isolation Kit, Exiqon
- i. Transfer 500 µl of serum into 1.5 ml microcentrifuge tube.
- ii. Add 120 µl Lysis Solution BF, vortex 5 sec, incubate for 3 min at room temperature.
- iii. Add 50 µl Protein Precipitation Solution BF, vortex 5 sec, incubate for 1 min at room temperature.
- iv. Centrifuge at 11,000g for 3 min at room temperature.
- v. Transfer clear supernatant to new microcentrifuge tube.
- vi. Add 675 µl Isopropanol, vortex 5 sec.
- vii. Assemble microRNA Mini Spin Column BF in a new collection tube and load 700 µl of sample into column.
- viii. Centrifuge at 11,000g for 30 sec at room temperature. Discard flow-through and return column to collection tube.
- ix. Load remaining sample onto column and centrifuge at 11,000g for 30 sec at room temperature. Discard flow-through and return column to collection tube.
- x. Add 700 µl Wash Solution 2 BF to column and centrifuge at 11,000g for 30 sec at room temperature. Discard flow-through and return column to collection tube.
- xi. Add 250 µl Wash Solution 2 BF to column and centrifuge at 11,000g for 2 min at room temperature. Discard flow-through and return column to collection tube.
- xii. Add 50 µl rDNase directly onto membrane of spin column.
- xiii. Close lid and incubate for 15 min at room temperature
- xiv. Add 100 µl Wash Solution 1 BF to column and centrifuge at 11,000g for 30 sec at room temperature. Discard flow-through and return column to collection tube.
- xv. Add 700 µl Wash Solution 2 BF to column and centrifuge at 11,000g for 30 sec at room temperature. Discard flow-through and return column to collection tube.
- xvi. Add 250 µl Wash Solution 2 BF to column and centrifuge at 11,000g for 2 min at room temperature.
- xvii. Transfer spin column into a new collection tube.
- xviii. Add 50 µl RNase free H2O directly onto the membrane of spin column.
- xix. Close lid and incubate for 1 min at room temperature.
- xx. Centrifuge at 11,000g for 1 min at room temperature.
- xxi. Discard spin column and store purified RNA at -70°C.
B. miRNeasy Mini Kit
- i. Transfer 500 µl of serum into 1.5 ml microcentrifuge tube.
- ii. Add 700 µl QIAzol Lysis Reagent, vortex 5 sec, incubate for 5 min at room temperature.
- iii. Add 140 µl chloroform, shake vigorously for 15 sec, incubate for 3 min at room temperature.
- iv. Centrifuge sample at 12,000g for 15 min at 4°C.
- v. Transfer the upper aqueous phase to a new microcentrifuge tube.
- vi. Add 500 µl 100% Ethanol, mix by pipetting.
- vii. Assemble RNAeasy Mini Column in a new collection tube and load entire sample onto column.
- viii. Centrifuge sample at 8,000g for 15 sec at room temperature. Discard flow-through and return column to collection tube.
- ix. Add 700 µl Buffer RPE to column and centrifuge at 8,000g for 15 sec at room temperature. Discard flow-through and return column to collection tube.
- x. Add 500 µl Buffer RPE to column and centrifuge at 8,000g for 2 min at room temperature. Discard flow-through and return column to collection tube.
- xi. Centrifuge column at 8,000 x g for 1 min at room temperature to dry membrane.
- xii. Transfer spin column into a new collection tube.
- xiii. Add 30 µl RNase free H2O directly onto the membrane of spin column.
- xiv. Close lid and centrifuge for 1 min at 8,000 x g at room temperature.
- xv. Discard spin column and store purified RNA at -70°C.
C. SeraMir Exosome RNA Purification Kit
- i. Transfer 500 µl of serum into 1.5 ml microcentrifuge tube.
- ii. Add 100 µl ExoQuick-TC, mix well, incubate for 30 min at 4°C.
- iii. Centrifuge sample at 13,000 rpm for 2 min at 4°C.
- iv. Remove supernatant, add 350 µl Lysis Buffer to EV, vortex 15 sec, incubate for 5 min at room temperature.
- v. Add 200 µl 100% Ethanol, vortex 10 sec.
- vi. Assemble Spin Column in a new collection tube and load 700 µl of sample onto column.
- vii. Centrifuge sample at 13,000 rpm for 1 min at room temperature. Discard flow-through and return column to collection tube.
- viii. Load remaining sample onto column and centrifuge at 13,000 rpm for 1 min at room temperature. Discard flow-through and return column to collection tube.
- ix. Add 400 µl Wash Buffer to column and centrifuge at 13,000 rpm for 1 min at room temperature. Discard flow-through and return column to collection tube.
- x. Add 400 µl Wash Buffer to column and centrifuge at 13,000 rpm for 1 min at room temperature. Discard flow-through and return column to collection tube.
- xi. Centrifuge column at 13,000 rpm for 2 min at room temperature to dry membrane.
- xii. Transfer spin column into a new collection tube.
- xiii. Add 30 µl Elution Buffer directly onto the membrane of spin column.
- xiv. Close lid and centrifuge at 2,000 rpm for 2 min at room temperature.
- xv. Increase speed and centrifuge at 13,000 rpm for 1 min at room temperature.
- xvi. Discard spin column and store purified RNA at -70°C.
Author information
Irene Yan & Tushar Patel, Patel Lab, Mayo Clinic
Correspondence to: Tushar Patel ([email protected])
Source: Protocol Exchange (2015) doi:10.1038/protex.2015.004. Originally published online 19 January 2015.