Isolate total cell RNA from six well plate for miRNA analysis
Shui Qing Ye (11/01/09)
1. Materials
1-1. miRNeasy Mini Kit (cat. no.217004, Qiagen)
1-2. Phase Lock Gel Heavy 2 ml ( cat.no.2302800, 5 Prime which can be ordered from Fisher)
2. Procedure
Things to do before starting
A. Thaw the cells, which were stored at -80C freezer in 700 µl QIAzol, at RT for 20 ~ 30 min.
B. Spin down phase lock gel tube at 16,000 × g, 1 min, RT in an Eppendorf microcentrifuge.
C. Buffers RWT and RPE are supplied as concentrates. Before using for the first time, add the
required amounts of ethanol (100%, molecular biology grade), as indicated on the bottle, to
obtain a working solution.
2-1. Transfer cell lysates in QIAzol lysis reagent to the prespun phase lock gel tubes.
2-2. Add 140 µl chloroform (molecular biology grade) and cap tube securely. Horizontally shake the
tubes vigorously in hands for 15 sec.
2-3.Centrifuge at 16,000 × g, 5 min, RT in an Eppendorf microcentrifuge.
2-4.Transfer upper aqueous phase about 400 µl to a new collection tube (1.5 ml size), add 600 µl ethanol
(100%, molecular biology grade), and mix thoroughly by pipetting up and down 5 times.
2-5. Transfer 500 µl above mixture into RNeasy Mini spin column in 2 ml collection tube. Close lid,
centrifuge at 16,000 × g, 15 sec, RT in an Eppendorf microcentrifuge by pressing Short key.
Discard flow-through.
2-6. Repeat step 2-5 using reminder of sample.
2-7. Pipet 500 µl Buffer RPE onto RNeasy Mini spin column in 2 ml collection tube. Close lid, centrifuge
at 16,000 × g, 15 sec, RT in an Eppendorf microcentrifuge by pressing Short key. Discard flowthrough.
2-8. Pipet another 500 µl Buffer RPE onto RNeasy Mini spin column in 2 ml collection tube. Close lid,
centrifuge at 16,000 × g, 2 min, RT in an Eppendorf microcentrifuge.
2-9. Transfer the RNeasy Mini spin column to a new 1.5 ml collection tube. Pipet 30 µl RNase-free water
directly onto the RNeasy Mini spin column Close lid, centrifuge at 16,000 × g, 1 min, RT in an
Eppendorf microcentrifuge.
2-10. Quantitate RNA and store them at -80C freezer.