-
Homogenization of
tissue:
-
For small
amounts ( 50 mg) of tissue, homogenization can be performed in a
microcentrifuge tube using the plastic pestle provided with this kit. The
microcentrifuge tube should be pre-chilled either in a -70°C freezer or
in a dry ice/ethanol bath. Weigh the tissue while frozen. Add the frozen
tissue to the tube. Add 1.0 mL of Lysis/Binding Reagent to the tube and
homogenize the tissue with the pestle. Continue until the tissue has been
completely homogenized. It may be necessary to pass the lysate through a
20-gauge syringe needle several times to completely disrupt the cells.
-
For large amounts (> 50
mg) of tissue, homogenization should be performed using a mechanical
homogenizer, although tissue can be prepared using liquid nitrogen and a
mortar/pestle followed by disruption with a 20-gauge syringe needle.
-
Regardless of the
homogenization method used, it is imperative that the tissue remains
frozen until homogenization is initiated. The tissue homogenate should
then be centrifuged for 1-2 minutes to pellet any cellular debris.
-
Maximum
binding capacity is 1.2 µg
mRNA per 100 µL
of Mag-Net Oligo dT Matrix. Adjust the amount of tissue homogenate used
per isolation accordingly.
-
mRNA Isolation
Protocol:
-
Remove the
Lysis/Binding Reagent from the pre-washed Mag-Net Oligo dT Matrix (Step 1)
using the Mag-Net Tube Stand to collect the magnetic particles to the side
of the tube while removing the supernatant.
-
Add
the cleared lysate (from Step 2) to the tube of pre-washed Mag-Net Oligo
dT Matrix and lightly vortex to resuspend. Mix the tube by inversion for
3-5 minutes at room temperature to allow annealing of the mRNA to the
Oligo dT Matrix. Place the tube in the Mag-Net Tube Stand for 2 minutes
and carefully remove the supernatant. It
may require more than two minutes to collect the magnetic particles
depending on the viscosity of the homogenate.
-
Wash the magnetic
matrix with 1.0 mL of the Extraction Reagent, resuspend completely, and
carefully remove all traces of the supernatant using the Mag-Net Tube
Stand. Repeat a second time.
-
Wash once using 1.0 mL
of Wash Reagent, resuspend completely, and remove all of the supernatant
utilizing the Mag-Net Tube Stand.
-
Add 20 µL of Elution
Reagent to the tube, gently vortex, and pulse centrifuge to pool the
magnetic particles and Elution Reagent to the bottom of the tube. Incubate
at 65°C for 2 minutes. Gently vortex and pulse centrifuge the tube, then
place the tube in the Mag-Net Tube Stand for 30 seconds. Transfer the
supernatant containing the mRNA to a sterile, RNase-free microcentrifuge
tube. The final elution step may also be performed using sterile,
deionized water or loading buffer containing formamide.
