SXZ / mRNA ISOLATION PROTOCOL
mRNA Isolation from total RNA with Oligotex mRNA mini Kit
(Qiagen, Cat# 70022)
Before starting:
-Heat Oligotex Suspension to 37°C in a water bath or heating block. Mix by vortexing,
and then place at room temperature.
- Heat a water bath or heating block to 70°C, and heat Buffer OEB.
-Bring the RNA samples (<250ug) to 250ul with RNase-free water
-Add 250ul of Buffer OBB, mix well
-Add 15ul of Oligotex suspension, mix well
-Incubate the samples at 70C for 3min.
-Incubate the samples at room temperature for 10-20min
-Centrifuge at max. speed for 2 min
- Remove the supernatant by pipetting
-Re-suspend the Oligotex-RNA pellet in 400ul buffer OW2 by pipetting
-Apply the Oligotex-RNA pellet to a spin colum
-Centrifuge for 1 min at Max speed
-Transfer the column to a new 1.5ml tub, add 400ul of OW2, and mix well by pipetting
-Centrifuge for 1 min at Max speed
-Transfer the column to a new 1.5ml tub,
-Apply 136ul of OEB buffer (70C) to the column, pipette up and down 5-8 time to resuspend the resin
-Centrifuge for 1 min at max. speed,
-Apply 136ul of OEB buffer (70C) again to the column, pipette up and down 5-8 time to
re-suspend the resin
-Centrifuge for 1 min at max. speed,
-For the two elution of total 272ul mRNA, to do precipitation by adding:
272ul
mRNA
1ul
Linear Acrylamine (5ug/ul)
27.2ul
3M sodium acetate , pH 5.2
900ul
100% ETOH
1200
-total volume should be 1.5ml
-Mix well and keep at -80C for overnight.
-Centrifuge the precipitation solution at 4C with max speed for 30min
-Washing the pellets with 70% ETOH
-Centrifuge at 4C with max speed for 30min
-Remove the residual ETOH
-Dry the pellets by 10min
-Re-suspend the pellets in 9.5ul RNase-free water.
-the mRNA is ready for cDNA library construction.