1.Strong magnetic response
2.High specificity
3.Simple operation
4.Suitable for high-throughput processing

Rapid and highly efficient isolation of intact, high-purity mRNA from eukaryotic total RNA or directly from animal/plant tissues and cell lysates.

User-Supplied Reagents

Note: All reagents should be prepared with DEPC-treated water and equilibrated to room temperature prior to use. If precipitates form, warm at 37°C for 10 min to dissolve.

1.Bead Pretreatment
(1)Resuspend the beads thoroughly by repeated inversion or gentle vortexing. Pipette the required volume of bead suspension into a fresh RNase-free microcentrifuge tube, add an equal volume of Binding Buffer, and vortex to resuspend.
(2)Place the tube on a magnetic separator and let it stand for 1 min to achieve magnetic separation. Aspirate and discard the supernatant, then remove the tube from the separator.
(3)Add a volume of Binding Buffer equal to the original bead suspension volume to resuspend the beads for downstream use.
Note: If isolating mRNA from total RNA, add half the original volume of buffer to concentrate the beads to 10 mg/mL.

2.mRNA Purification from Total RNA (Taking 100 µg total RNA as an example)
(1)Adjust the total RNA volume to 100 µL with DEPC-treated water, then add an equal volume (100 µL) of Binding Buffer.
(2)Heat at 65°C for 2 min to disrupt RNA secondary structures, then immediately transfer the sample onto ice to cool.
(3)Add the 200 µL total RNA solution to 100 µL of pre-washed Oligo (dT) Mag Beads, and mix thoroughly.
Note: Use 1 mg of washed beads resuspended in 100 µL of Binding Buffer per 100 µg of total RNA (refer to "1. Bead Pretreatment").
(4)Incubate on a rotary mixer at room temperature for 10 min. Perform magnetic separation, aspirate and discard the supernatant, then remove the tube.
(5)Add 200 µL of Washing Buffer B, mix gently, perform magnetic separation, aspirate and discard the supernatant, and remove the tube. Repeat this wash step once.
Note: Ensure the beads are completely resuspended during each wash step, and remove as much supernatant as possible after the second wash.
(6)Choose one of the following procedures based on downstream requirements:
a)If elution of mRNA from beads is NOT required: Wash the beads once more with the enzymatic reaction buffer required for the downstream experiment.
b)If elution of mRNA is required: Carefully and completely remove Washing Buffer B (take care not to aspirate beads). Add 10–20 µL of Nuclease-Free Water or 10 mM Tris-HCl (pH 7.5), mix thoroughly, and incubate at room temperature for 2 min. Perform magnetic separation and transfer the mRNA-containing supernatant to a fresh RNase-free tube.
Note: To increase elution efficiency, heating at 65°C–75°C during elution is recommended.

3.mRNA Isolation from Cell Lysates
(1)Wash the cell suspension with PBS and centrifuge to collect the cell pellet. The cell pellet can be used immediately or snap-frozen in liquid nitrogen and stored at -80°C.
(2)Add 1 mL of Lysis/Binding Buffer to the cell pellet containing 1-4 x 10^6 cells, and pipette up and down several times to ensure complete cell lysis. An increase in lysate viscosity due to DNA release indicates thorough cell lysis.
(3)Perform a shear step to reduce lysate viscosity: Pass the lysate 3 times through a 21-gauge needle attached to a 1–2 mL syringe.
Note: Repeated shearing may generate slight foaming; brief centrifugation for 30 s will eliminate the foam without affecting mRNA yield.
(4)The lysate can be used immediately for mRNA extraction or frozen at -80°C for later use.
(5)Place the pre-washed Oligo (dT) Mag Beads on the magnetic rack to discard the supernatant. Add the cell lysate to the beads and mix thoroughly.
(6)Rotate at room temperature for 5 min to promote mRNA binding; extend incubation time appropriately if the solution is viscous. Perform magnetic separation, aspirate and discard the supernatant, and remove the tube.
(7)Wash the beads twice: Wash once with 1 mL of Washing Buffer A, followed by one wash with 1 mL of Washing Buffer B. Ensure complete resuspension of beads during washing and complete removal of supernatant after each wash.
(8)Choose one of the following procedures based on downstream requirements:
a)If elution of mRNA is NOT required (e.g., for downstream solid-phase cDNA synthesis): Wash the beads once with 500 µL of Washing Buffer B, then wash once with the enzymatic reaction buffer required for downstream assays.
b)If elution of mRNA is required: Carefully and completely remove Washing Buffer B (take care not to aspirate beads). Add 10–20 µL of Nuclease-Free Water or 10 mM Tris-HCl (pH 7.5), mix thoroughly, and incubate at 65°C–75°C for 2 min. Perform magnetic separation and transfer the mRNA-containing supernatant to a fresh RNase-free microcentrifuge tube.
Note: mRNA content varies among different tissues and cell types. Mammalian cells typically contain ~10–30 pg of total RNA per cell, of which mRNA accounts for approximately 1%–5%.

4°C, 2 years.

1.Do not freeze the magnetic beads. Ensure the beads remain submerged in storage buffer at all times to prevent drying.
2.Ensure magnetic separation duration is at least 1 min per step to avoid bead loss.
3.Resuspend beads thoroughly by vortexing prior to pipetting. Avoid generating bubbles.
4.Use high-quality low-binding pipette tips and microcentrifuge tubes to prevent bead loss due to wall adhesion.
5.Users may collect supernatants from antibody- or antigen-binding steps to evaluate binding efficiency via Western blot or SDS-PAGE.
6.Binding affinities may vary across different antibodies and antigens in IP experiments. If optimal results are not obtained using the provided buffer system, users may optimize custom buffer conditions.
7.This product is restricted for use by professional personnel for scientific research purposes only. It must not be used for clinical diagnosis or treatment, food or drugs, and must not be stored in residential areas.
8.For your safety and health, please wear a lab coat and disposable gloves during operations.