Dynabeads® mRNA DIRECT Micro Kit - mRNA isolation for RT-PCR amplification
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实验原理
Use the Dynabeads® mRNA DIRECT™ Micro Kit for simple and rapid isolation of mRNA from small cell and tissue samples, for direct use in reverse transcription PCR. The direct mRNA isolation is completed in only 15 minutes, with no need for preliminary total RNA purification. The oligo (dT) bound to the bead surface is used both to capture the mRNA and as a primer for the reverse transcriptase to synthesise the first strand cDNA.
实验试剂
实验设备
Dynal® magnet: see www.invitrogen.com/magnets
Sterile, RNase-free micro centrifuge tubes
Sterile, RNase-free pipette tips
实验步骤
1. Preparation of Dynabeads Oligo (dT)25
1) Resuspend the Dynabeads thoroughly before use.
2) Transfer Dynabeads needed for all samples (using 20 μl Dynabeads per mRNA isolation) from the stock tube suspension, to an RNase-free microcentrifuge tube.
3) Place the tube on a Dynal magnet.
4) After 30 seconds (or when the suspension is clear) remove the supernatant.
5) Remove the tube from the magnet and pre-wash Dynabeads by resuspending in Lysis/Binding Buffer to the original volume by pipetting.
6) Place the tube on the magnet and remove supernatant.
Note: Do not allow the Dynabeads to dry, as this may lower their capacity.
7) Remove the tube from the magnet and resuspend the beads in Lysis/binding buffer to the original volume. Aliquot 20 μl suspension to each sample tube.
2. Preparation of lysate from cultured cells and cell suspensions
This protocol is recommended for samples containing up to 1 x 104 cultured cells or up to 2.5 x 104 mononuclear cells.
1) Wash the cell suspension in phosphate-buffered saline (PBS) prior to preparing a cell pellet by centrifugation. The cell pellet can be used immediately, or frozen in liquid nitrogen and stored at -80°C for later use.
2) Add 100 μl Lysis/Binding Buffer to the fresh/frozen cell pellet. Perform a repeated passage of the solution through a pipette tip to obtain complete lysis. The lysate may be frozen (-80°C) and stored for later use.
3) Combine the lysate with Dynabeads Oligo (dT)25 in the mRNA isolation protocol below, step 2.C.
3. Protocol for mRNA isolation for PCR amplification
1) Prepare the Dynabeads and the lysate.
2) Transfer the clear lysate to the tube containing 20 μl pre-washed Dynabeads.
3) Mix by pipetting up and down a few times.
4) Place the tube on a sample mixer or roller for 5 min. at room temperature to allow the mRNA to anneal to the Dynabeads with continuous rotation.
5) Place the sample tube on the magnet and discard the supernatant.
6) Remove the sample tube from the magnet and resuspend the Dynabeads-mRNA complex in 100 μl Washing Buffer A by careful pipetting.
7) Place the sample tube on the magnet and discard the supernatant.
9) Resuspend the Dynabeads-mRNA complex in 100 μl Washing Buffer B.
10) Transfer the suspension to a new tube.
11) Place the new sample tube on the magnet and discard the supernatant.
12) Resuspend the Dynabeads-mRNA complex in 100 μl Washing Buffer B.
13) Place the sample tube on the magnet and discard the supernatant.
14) Remove the sample tube from the magnet and resuspend in 100 μl of ice-cold 10mM Tris-HCl.
15) Place the sample tube on ice prior to PCR amplification. We recommend preparing the reverse transcription PCR mix before starting the mRNA isolation protocol.
16) Immediately before adding the reverse transcription PCR mix, place the tube on the magnet and discard the supernatant.
17) For one-tube PCR, resuspend the Dynabeads-mRNA complex in 50 μl reverse transcritption PCR mix and transfer to PCR-tube. For two-step PCR, resuspend the Dynabeads-mRNA complex in the reverse tran-scription PCR reaction mix according to the manufacturers recommendation.
18) Perform cDNA synthesis as recommended by the manufacturer of the reverse transcriptase. When using a thermostable reverse transcriptase and the bead-bound oligo (dT) as primer for first strand cDNA synthesis, an initial incubation at 50°C for 5 minutes is necessary before proceeding at the recommended temperature.
注意事项
1. Keep the vials of Dynabeads Oligo (dT)25 in an upright position to ensure that the beads are covered with buffer, as drying will reduce their performance. Should the Dynabeads Oligo (dT)25 by accident have dried, the beads can be resuspended in the buffer they are supplied in by placing the vial on a roller or equivalent overnight (4°C). This treatment will restore their complete functionality.
2. Wear disposable gloves and change them frequently.
3. Use sterile, RNase-free microtubes and pipette tips.
4. RNase inhibitors may be added to the protocol at any step.
