RNA Isolation for Array Screening (RNA Later Method)
October 09, 2000
[Use RNase-free eppendorf tubes & tips]
1. Quickly! transfer 4-7 mls of cell culture to a 14 ml Falcon round
bottom tube (Cat# 352059) containing an equal volume RNA later solution
(Ambion cat# 7021) on ice.
2. Store sample (with RNA later) at 4oC for up to 6 hrs (RNA decay
is observed after 6 hrs) (It is best if RNA is isolated immediately).
3. Pellet cells at 4oC using the Allegra 21R Beckman centrifuge
and F0630 rotor (rotor pre-cooled and stored at 4oC), spin for 15-30
min at 10,000 rpm (longer times for larger volume harvests). Note:
RNA Later prevents cells from pelletting well, use care when decanting supernatant.
5. Carefully pull off supernatant with glass pipet and resuspend pellet in
200 ul ice-cold lysis solution
(lysozyme-containing TE as described in Qiagen RNeasy Kit).
6. Follow RNeasy protocol from Qiagen (If harvest O.D. was over 0.3 then split
sample and use 2x Qiagen solutions and 2 columns). Note: You will over-load
the columns if you don’t!
7. Collect final eluents from RNeasy preparation and combine duplicate samples
into one 1.5 ml epp-tube and speed-vac sample (at 45 or 60oC) until
it is almost completely dry. If it does happen to completely dry out that is
OK, Resuspend RNA in 10-50 ul RNA
free H2O.
8. Determine RNA concentration by measuring A260.
9. Storage of RNA samples: Add 2 volumes of 100% ethanol to RNA
in water. Store RNA in ethanol at –80oC.
10. Mix well before removing aliquot. Remove required volume and
precipitate, wash, then proceed as usual.
Lysis Solution:
10mM Tris pH 8.0
1mM EDTA pH 8.0
-Must be RNase free solutions
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