P-CAGE-25053 - P-CAGE-25053
Each samples are preserved with grinding ball in 2-ml tube and stored at the deep freezer. Total RNA was isolated using RNeasy Kit (QIAGEN).
1. Grind sample under liquid nitrogen to a fine powder using Mixer Mill MM 301(Retsch).
2. Add 450 µl of Buffer RLT (1% 2-mercaptoethanol) to a maximum of 100 mg of tissue powder. Vortex vigorously.
3. Apply lysate to the QIAshredder spin column sitting in a 2-ml collection tube, and centrifuge for 2 min at maximum speed. Transfer flow-through fraction from QIAshredder to a new tube without disturbing the cell-debris pellet in the collection tube.
4. Add 0.5 volumes 100% ethanol to the cleared lysate and mix well by pipetting.
5. Apply sample including any precipitate which may have formed, onto an RNeasy mini spin column sitting in a 2-ml collection tube. Centrifuge for 15 sec at 8000 x g.
6. Pipet 700 µl Buffer RW1 onto the RNeasy column, and centrifuge for 15 sec at 8000 x g to wash.
7. Transfer RNeasy column into a new 2-ml collection tube. Pipet 500 µl Buffer RPE onto the RNeasy column, and centrifuge for 15 sec at 8000 x g.
8. Add 500 µl Buffer RPE to the RNeasy column, and centrifuge for 2 min at maximum speed to dry the RNeasy membrane.
9. Transfer RNeasy column into a new 1.5-ml collection tube, and pipet 30 of RNase-free water directly onto the RNeasy membrane. Centrifuge for 1 min at 8000 x g.
Amplification, Extracted product
|All experiments using protocol P-CAGE-25053: (E-ATMX-6, E-ATMX-7)|