General RNA purification protocol using magnetic beads

This protocol assumes that you start with a homogenized sample containing a mixture of a desired RNA and other undesired compounds. The sample was previously lyzed or processed, so RNA is free floating in the solution.

Materials

• Sample to extract RNA from
• Sergi Lab Supplies magnetic beads for RNA purification
• Freshly made 80% ethanol solution
• Nuclease-free water
• Pipette tips with filters, nuclease-free
• Nuclease remover solution to spray pipettes, bottles and surfaces

Equipment

• Microcentrifuge tubes (PCR, 0.6 mL, 1.5 mL or larger)
• Magnetic rack for those tubes
• Vortex mixer

Preparation

1. Clean up your workbench to minimize the chance of RNAse contamination. Wipe with water, 70% ethanol. Then, wipe all the surfaces with a nuclease remover solution of your choice. Then, wipe with nuclease-free water to remove any residual chemicals.
2. Treat similarly all pipettes and reagents bottles you are going to use
   
3. Prepare fresh 80% ethanol solution. In a 15 mL tube, mix 8 mL of ethanol and 2 mL of nuclease-free water. Vortex.
4. Vortex a bottle with magnetic beads thoroughly (at least 30 seconds). 
5. Ensure your sample occupies less than 1/4 volume of the test tube, so there is enough space to add magnetic beads

RNA absorption on beads

1. Add RNA magnetic beads to the sample. For the whole RNA extraction, add 2-3x of a sample volume. For example, if the sample volume is 50 uL, add 100 uL of magnetic beads suspension. For selectively extracting larger molecules of RNA, separating them from smaller molecules, add 0.6-1x of the sample volume (the RNA yield will likely decrease)
   Refer to the following picture to estimate the desired beads to sample ratio:
   
   
2. Vortex thoroughly for several seconds. Ensure the suspension is homogenous.
3. Leave the sample with beads on bench for 10 minutes, so the RNA get attached to the beads
4. Place the tube with the sample on the magnetic rack. Wait 1-3 minutes for solution to clear from magnetic beads
5. Discard the supernatant. The RNA is now on beads

Washing

Perform two washing cycles with 80% ethanol

1. Keep the sample tube on the magnetic rack
2. Add 200-500 uL of the 80% ethanol to the beads
3. Wait for 30 seconds
4. Remove ethanol solution
5. Repeat one more time
6. Using a 20 uL pipette, remove the remaining ethanol solution from the bottom of the tube
7. Leave the tube open for 3-4 minutes so the remaining ethanol evaporates. Do not overdry, as the RNA yield will decrease significantly.

Elution

1. Remove the tube from the magnetic rack
2. Add 30-50 uL of a nuclease-free water
3. Vortex thoroughly so the pellet fully resuspends
4. Leave the tube on bench for 10 minutes for the elution to finish
5. Place the tube on the magnetic rack. Wait for beads to pull to the side
6. Transfer the supernatant that contains the desired RNA to a new tube.
7. Discard the tube with beads.