Circulating miRNA Extraction: A Method to Isolate miRNA from Plasma Samples Via Organic Extraction and Small RNA Enrichment

NOTE: Perform all of the following steps under a sterilized fume hood according to Good Laboratory Practice (GLP).

1. Plasma Collection and Storage

1. Collect 3 mL of peripheral blood sample in a K3E EDTA tube.
2. Centrifuge the tube at room temperature at 1,880 x g for 15 min.
3. Recover the supernatant plasma in aliquots of 450 µL.
4. Store the samples at -80 °C until use.
   NOTE: Process the peripheral blood tube within 2 h of collection. When recovering the plasma from the tube, do not disturb the lymphocyte layer.

2. Extraction of Circulating miRNAs from Plasma Samples

1. Prepare all the required solutions and thaw the samples for the extraction steps.
   1. Add 375 µL of 2-mercaptoethanol to the 2x denaturing solution and mix well.
   2. Add 21 mL of ACS grade 100% ethanol to the miRNA wash solution I and mix well.
   3. Add 40 mL of ACS grade 100% ethanol to the wash solution 2/3 and mix well.
   4. Allow a plasma aliquot to thaw at room temperature and then begin the extraction steps.

2. Organic extraction
   1. Transfer 400 µL of the plasma sample into an RNase-free 2 mL tube.
   2. Add 400 µL of 2x denaturing solution, mix by vortexing and briefly centrifuge.
   3. Add 4 µL of 1 nM spike-in, mix by vortexing and briefly centrifuge.
   4. Add 800 µL of acid phenol-chloroform.
   5. Vortex for 60 s and centrifuge at maximum speed (≥10,000 x g) for 15 minutes.
   6. Transfer the upper aqueous phase to a fresh tube. Do not disturb the interphase. Note the volume recovered and discard the tube containing the non-aqueous phase.
      NOTE: The 2x denaturing solution is stored at 4 °C and may solidify at this temperature. Before use, warm the solution to 37 °C, occasionally agitating for 10-15 min. Be careful to withdraw the bottom phase containing the acid phenol-chloroform, not the aqueous buffer that lies on top of the mixture. Preheat the elution solution or nuclease-free water to 95 °C. Be careful to heat an adequate volume of solution (100 µL per sample + 10% excess).

3. Small RNA enrichment
   1. Add 1/3 volume of ethanol 100% to the aqueous phase from step 2.2.6 and mix thoroughly.
   2. Place a filter cartridge into a fresh collection tube for each sample.
   3. Transfer up to 700 µL of lysate from step 2.3.1 and ethanol into the filter cartridge and centrifuge at 10,000 x g for 30 s.
   4. If there is >700 µL of mixture left, place the filtrate in a fresh tube and repeat step 2.3.3; repeat until the entire mixture has passed through the filter cartridge.
   5. Measure the total volume of the flow-through.
   6. Add 2/3 volume of ethanol 100% to filtrate and mix thoroughly.
   7. Place a second filter cartridge in a fresh collection tube.
   8. Transfer up to 700 µL of sample volume into the cartridge and centrifuge at 10,000 x g for 30 s. Discard the flow-through.
   9. Repeat step 1.3.8 until all the mixture has passed through the filter cartridge.
   10. Apply 700 µL of miRNA wash solution 1 to the filter cartridge and centrifuge the filter cartridge with the collection tube at 10,000 x g for 15 s. Discard the flow-through. Place the filter cartridge in the same collection tube.
   11. Apply 500 µL of wash solution 2/3 to the filter cartridge and centrifuge the filter cartridge with the collection tube at 10,000 x g for 15 s. Discard the flow-through. Place the filter cartridge in a fresh collection tube.
   12. Place the filter cartridge in a fresh collection tube and repeat step 2.3.11 with 500 µL of wash solution 2/3. Discard the flow-through and transfer the filter cartridge to a fresh collection tube.
   13. Centrifuge the tubes with the filter cartridges at 10,000 x g for 1 min.
   14. Transfer the filter cartridge into a fresh 1.5 mL collection tube and add 100 µL of preheated (95 °C) elution solution or nuclease-free water.
   15. Centrifuge at 10,000 x g for 30 s to recover miRNAs and store at -80 °C.
       NOTE: A white precipitate may form in the wash solution 2/3. This precipitate is excess EDTA released from the solution. Avoid drawing up these crystals during the pipetting steps.