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 donít!
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
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 Ė80oC.
10. Mix well before removing aliquot. Remove required volume and
precipitate, wash, then proceed as usual.
10mM Tris pH 8.0
1mM EDTA pH 8.0
-Must be RNase free solutions