Oligo(dT) Magnetic Beads

1. Prepare the cells or tissue lysate.
2. Prepare the Oligo(dT)30 Magnetic Beads.
3. Add the lysate to the prepared beads, incubate and wash the beads.
4. Elute the bound mRNA and proceed for further analysis.

Note: Please note that the protocol described is a general guideline and should be optimized based on individual experimental needs.

1. Pellet the cells (1-10×10⁶), by centrifugation (e.g., 300-400 × g for 10 minutes at 4°C).
2. Resuspend the cells in phosphate buffer salt (PBS) and centrifuge again. The pellet is used immediately after centrifugation.
3. Add the appropriate volume (1mL) of lysis/binding buffer to either a frozen cell pellet or to a fresh cell pellet.

1. Divide the tissue equally during freezing to avoid mRNA degradation.
   

   Note: Use appropriate amount (plant tissue ~100mg, animal tissue ~50mg), because the use of too much tissue will reduce mRNA yield and purity.

2. Grind the frozen tissue rapidly in liquid nitrogen.
3. Transfer the frozen powder to an appropriate volume (1mL) of lysis/binding buffer and homogenize until it is completely dissolved. Rapid dissolution in binding buffer is essential to prevent mRNA degradation.

1. Resuspend the oligo (dT)30 beads completely before use.
2. Transfer the desired volume (50μL-1mL) of beads from the stock tube to an RNase-free 1.5-mL microcentrifuge tube and place the tube on a magnet block.
3. When the suspension is clear, remove the supernatant.
4. Remove the tube from the magnet and add the same amount of fresh lysis buffer to clean the magnetic beads. 
   Note: For very small bead volumes (50–100 μL) use 100 μL wash volume to ease handling.
   

1. Lysis/Binding Buffer

2. Washing Buffer A

3. Washing Buffer B

4. Elution Buffer

1. Remove the lysis buffer from the pre-washed Oligo(dT)30 beads by placing on the magnet until the suspension is clear and discard the supernatant.
2. Remove the microcentrifuge tube from the magnet and add the appropriate volume of sample lysate (500μL-1mL).
3. Resuspend the beads completely in the sample lysate.
4. Incubate with continuous mixing (rotating or roller mixer) for 3mins at room temperature to allow the polyA tail of the mRNA to hybridize to the oligo (dT)30 on the beads.
   

   Note: If the solution is viscous, increase the incubation time or adjust the volume appropriately.

5. Place the vial on the magnet for 2 minutes and remove the supernatant. If the solution is noticeably viscous, increase this time to ~10 minutes.
6. Wash the beads/mRNA complex two times with the appropriate volume (1mL) of washing buffer A at room temperature.
7. Use the magnet to separate the beads from the solution between each washing step.
8. Wash the beads/mRNA complex once with the appropriate volume（1mL) of washing buffer B at room temperature. Use the magnet to separate the beads from the solution.
9. Add an appropriate volume (20-100μL) of elution buffer and incubate at 65°C to 80°C for 2 minutes.
10. Immediately place the tube on the magnet, transfer the supernatant containing the mRNA to a new RNase-free tube and place this tube on ice. Proceed further with downstream analysis.