Isolation Purification and Separation Cell Biology Molecular Biology

scientificprotocols authored about 3 years ago

Authors: Irene Yan & Tushar Patel

Abstract

The study of extracellular RNA has been recently reported as an important mechanism of intercellular signaling. Extracellular vesicles which contain protein, mRNA, and non-coding RNA.can be isolated from cell culture supernatant. Extracellular vesicle RNA isolated from tumor cells in culture can be a useful tool for analyzing the role of either extracellular vesicles or extracellular vesicle RNA. This protocol presents a method of isolating extracellular vesicle RNA from human cholangiocarcinoma cells in culture.

Reagents

  1. One of the following commercial kits:
    • miCURY RNA Isolation Kit –Biofluids (Exiqon, #300112)
    • miRNeasy Mini Kit (Qiagen, #217004)
    • SeraMir Exosome RNA Purification Kit (System Biosciences, #RA806TC-1)
  2. Isopropanol
  3. Absolute ethanol
  4. Nuclease free H2O

Equipment

  1. Bench top centrifuge
  2. Vortex Mixer
  3. Micropipettes with sterile tips 20-1000
  4. Sterile collection bottle
  5. Microcentrifuge tubes
  6. 0.22 µm PES vacuum filter

Procedure

  1. Grow KMBC cholangiocarcinoma cells on 32×10 cm tissue culture plates. Use Extracellular vesicle (EV)-cleared media for cultures (total 400 ml). Incubate on cells for 48 hr.
  2. Collect supernatant and filter through a 0.22 µM PES filter
  3. Aliquot into ultracentrifugation tubes, and centrifuge for 70 min at 100,000 x g at 4°C.
  4. Discard supernatant, and resuspend pellet from each tube with 1 ml PBS.
  5. Combine washed pellets into one ultracentrifuge tube, and centrifuge for 70 min at 100,000 x g at 4°C.
  6. Discard supernatant, resuspend final EV pellet with 600 µl PBS. Store samples at -70°C until ready for use.
  7. RNA Isolation can be performed from one of the following options.

A. miCURY RNA Isolation Kit, Exiqon

  • i. Transfer 200 µl of EV into 1.5 ml microcentrifuge tube.
  • ii. Add 60 µl Lysis Solution BF, vortex 5 sec, incubate for 3 min at room temperature.
  • iii. Add 20 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 270 µl Isopropanol, vortex 5 sec.
  • vii. Assemble microRNA Mini Spin Column BF in a new collection tube and load entire sample onto column.
  • viii. Centrifuge at 11,000g for 30 sec at room temperature. Discard flow-through and return column to collection tube.
  • ix. 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.
  • x. 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.
  • xi. Add 50 µl rDNase directly onto membrane of spin column.
  • xii. Close lid and incubate for 15 min at room temperature
  • xiii. 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.
  • xiv. 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.
  • xv. Add 250 µl Wash Solution 2 BF to column and centrifuge at 11,000g for 2 min at room temperature.
  • xvi. Transfer spin column into a new collection tube.
  • xvii. Add 50 µl RNase free H2O directly onto the membrane of spin column.
  • xviii. Close lid and incubate for 1 min at room temperature.
  • xix. Centrifuge at 11,000g for 1 min at room temperature.
  • xx. Discard spin column and store purified RNA at -70°C.

B. miRNeasy Mini Kit

  • i. Transfer 200 µl of EV 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 (Since EV is already isolated by ultracentrifugation, omit ExoQuick-TC precipitation step)

  • i. Transfer 200 µl of EV into 1.5 ml microcentrifuge tube.
  • ii. Add 140 µl Lysis Buffer to EV, vortex 15 sec, incubate for 5 min at room temperature.
  • iii. Add 80 µl 100% Ethanol, vortex 10 sec.
  • iv. Assemble Spin Column in a new collection tube and load entire sample onto column.
  • v. Centrifuge sample at 13,000 rpm for 1 min at room temperature. Discard flow-through and return column to collection tube.
  • vi. 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.
  • vii. 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.
  • viii. Centrifuge column at 13,000 rpm for 2 min at room temperature to dry membrane.
  • ix. Transfer spin column into a new collection tube.
  • x. Add 30 µl Elution Buffer directly onto the membrane of spin column.
  • xi. Close lid and centrifuge at 2,000 rpm for 2 min at room temperature.
  • xii. Increase speed and centrifuge at 13,000 rpm for 1 min at room temperature.
  • xiii. Discard spin column and store purified RNA at -70°C.

Associated Publications

  1. Extracellular vesicle-mediated transfer of long non-coding RNA ROR modulates chemosensitivity in human hepatocellular cancer, Kenji Takahashi, Irene K. Yan, Takayuki Kogure, Hiroaki Haga, and Tushar Patel, FEBS Open Bio 4 () 458 - 467 doi:10.1016/j.fob.2014.04.007
  2. Involvement of Extracellular Vesicle Long Noncoding RNA (linc-VLDLR) in Tumor Cell Responses to Chemotherapy, K. Takahashi, I. K. Yan, J. Wood, H. Haga, and T. Patel, Molecular Cancer Research 12 (10) 1377 - 1387 01/10/2014 doi:10.1158/1541-7786.MCR-13-0636
  3. Modulation of hypoxia-signaling pathways by extracellular linc-RoR, K. Takahashi, I. K. Yan, H. Haga, and T. Patel, Journal of Cell Science 127 (7) 1585 - 1594 01/04/2014 doi:10.1242/jcs.141069
  4. Intercellular nanovesicle-mediated microRNA transfer: A mechanism of environmental modulation of hepatocellular cancer cell growth, Takayuki Kogure, Wen-Lang Lin, Irene K. Yan, Chiara Braconi, and Tushar Patel, Hepatology 54 (4) 1237 - 1248 29/07/2011 doi:10.1002/hep.24504

Author information

Irene Yan & Tushar Patel, Patel Lab, Mayo Clinic

Correspondence to: Tushar Patel ([email protected])

Source: Protocol Exchange (2015) doi:10.1038/protex.2015.006. Originally published online 26 January 2015.

Average rating 0 ratings