Oligo (dT)₃₀ Magnetic Beads

Recommended Reagents (Not Provided)

Magnetic Bead Pretreatment

Thoroughly resuspend the magnetic beads. Transfer 100 μL of magnetic beads into a 1.5 mL EP tube, add 1 mL of Binding Buffer, and resuspend the beads. Perform magnetic separation and discard the supernatant. Repeat this procedure 3-4 times. Finally, add 1 mL of Binding Buffer, resuspend the beads, and keep them ready for use.

RNA Extraction from Animal/Plant Tissues or Cells (For Reference Only)

1. Sample Pretreatment

1.1 Animal/Plant Tissues

Collect an appropriate amount of tissue and grind it thoroughly in liquid nitrogen. Transfer the ground tissue to a new EP tube. Add 1 mL of Lysis/Binding Buffer per 20-50 mg of animal tissue or per 100 mg of plant tissue. Homogenize thoroughly and incubate on a rotator at room temperature for 5 min. Centrifuge at 14,000 rpm for 5 min at room temperature and collect the supernatant. The supernatant may be used for subsequent mRNA purification or stored at -80°C.

Note: During lysis, repeatedly pipette the sample through a 1 mL syringe needle or pipette tip to shear genomic DNA and reduce the viscosity of the solution.

1.2 Cell Suspensions

Centrifuge the cell suspension at 4,000 rpm for 5 min at room temperature. Discard the supernatant and retain the cell pellet. Add 1 mL of Lysis/Binding Buffer per 1-4 × 10⁶ cells and pipette several times to ensure complete cell lysis. Centrifuge at 14,000 rpm for 5 min at room temperature and collect the supernatant. The supernatant may be used for subsequent mRNA purification or stored at -80°C.

Note: During lysis, repeatedly pipette the sample through a 1 mL syringe needle or pipette tip to shear genomic DNA and reduce the viscosity of the solution.

2. mRNA Extraction

2.1 Resuspend the pretreated magnetic beads, perform magnetic separation, and discard the supernatant.

2.2 Add the lysate to the magnetic beads and vortex for 3-5 min. Perform magnetic separation and discard the supernatant.

2.3 Wash the magnetic beads sequentially with 1 mL of Washing Buffer A and 1 mL of Washing Buffer B. Perform magnetic separation and discard the supernatant. Repeat the washing procedure 3-4 times to remove potential contaminants.

2.4 If the magnetic bead-mRNA complex will be used in a downstream enzymatic reaction, such as solid-phase cDNA synthesis, wash once with 500 μL of Washing Buffer B and then once with the appropriate enzyme reaction buffer before proceeding with downstream analysis.

2.5 To elute mRNA from the magnetic beads, add 10-20 μL of 10 mM Tris-HCl and incubate at 75-80°C for 2 min. After magnetic separation, immediately transfer the supernatant to a new RNase-free EP tube.

Purification of mRNA from Total RNA (For Reference Only)

The following procedure is described using 200 μg of total RNA as an example.

1. Mix 100 μL of sample containing 200 μg of total RNA with 100 μL of Binding Buffer.

Note: Total RNA may be diluted to 200 μg/100 μL using DEPC-treated sterile water or 10 mM Tris-HCl, pH 7.5.

2. Incubate at 65°C for 2 min and immediately place the sample on ice.

3. Mix the resulting 200 μL reaction solution with 100 μL of pretreated magnetic beads. Incubate on a rotator at room temperature for 5 min. Perform magnetic separation and discard the supernatant.

4. Wash the magnetic beads sequentially with 1 mL of Washing Buffer A and 1 mL of Washing Buffer B. Perform magnetic separation and discard the supernatant. Repeat the washing procedure 3-4 times to remove potential contaminants.

5. If the magnetic bead-mRNA complex will be used in a downstream enzymatic reaction, such as solid-phase cDNA synthesis, wash once with 500 μL of Washing Buffer B and then once with the appropriate enzyme reaction buffer before proceeding with downstream analysis.

6. To elute mRNA from the magnetic beads, add 10-20 μL of 10 mM Tris-HCl and incubate at 75-80°C for 2 min. After magnetic separation, immediately transfer the supernatant to a new RNase-free EP tube.