1. Transfer 200 µl of serum or plasma into a 1.5 ml microfuge tube.
  2. Add 200 µl of buffer XBP.
  3. Invert 5 times.
  4.  Add the sample/XBP mixture onto the exoEasy spin column.
  5. Centrifuge for 1 minute at 500 xg at room temperature.
  6. Discard flow-through.
  7. Add 10 ml of buffer XWP to the exoEasy spin column.
  8. Centrifuge for 5 minutes at 5,000 xg at room temperature.
  9. Transfer the spin column to a fresh collection tube.
  10. Add 700 μl Qiazol to the membrane of the spin column.
  11. Centrifuge for 5 minutes at 5,000 xg at room temperature.
  12. Transfer the flow-though, which is the lysate, to a PLG tube.<sup>1</sup>
  13. Vortex for 5 seconds.<sup>2</sup>
  14. Incubate at room temperature for 5 minutes.
  15. Add 90 µl chloroform.
  16. Shake vigorously for 15 seconds.
  17. Incubate for 3 min at room temperature.
  18. Centrifuge sample for 5 min at 12,000 x g at 4°C.
  19. Transfer the upper aqueous phase to a new microcentrifuge tube. 
  20. Carefully measure the aqueous phase and add 2 volumes of 100% ethanol.
  21. Mix gently and thoroughly. Do not centrifuge and do not delay moving on to the next step.
  22. Assemble a MinElute spin column in a new collection tube.
  23. Load up to 700 µl of the mixture from step 5, including any precipitate that may have formed, onto the column. 
  24. Centrifuge for 15 seconds at 1,000 x g at room temperature.<sup>3</sup> 
  25. Discard flow-through.
  26. Repeat steps 23-25 until entire sample has been loaded.
  27. Add 700 μl of buffer RWT to the RNeasy MinElute spin column. 
  28. Centrifuge for 15 seconds at 8000 x g at room temperature to wash the column. 
  29. Discard the flow-through. 30. Pipet 500 μl Buffer RPE onto the RNeasy MinElute spin column. 
  31. Centrifuge for 15 seconds at 8000 x g to wash the column. 
  32. Discard the flow-through.
  33. Pipet 500 μl Buffer RPE onto the RNeasy MinElute spin column. 
  34. Centrifuge for 2 minutes at ≥ 8000 x g \(≥10,000 rpm) at room temperature to wash the column. 
  35. Discard the collection tube with the flow-through.<sup>4</sup>
  36. Transfer the RNeasy MinElute spin column into a new 2 ml collection tube \(supplied). 
  37. Open the lid of the spin column, and centrifuge at full speed \(14,000 xg) for 5 min to dry the membrane.<sup>5</sup> 
  38. Discard the collection tube with the flow-through.<sup>4</sup> 
  39. Transfer the RNeasy MinElute spin column into a new 1.5 ml collection tube \(supplied). Add 14 μl RNase-free water directly to the center of the spin column membrane.<sup>6</sup> 
  40.  Centrifuge for 1 minute at full speed to elute the RNA.