Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 136

13. Add 500 l buffer RPE to the RNeasy spin column. Close the

lid gently and centrifuge for 2 min at ³8,000× g (³10,000 rpm)
to wash the spin column membrane. Discard the fl ow-through.
Notes: After centrifugation, carefully remove the RNeasy spin
column from the collection tube so the column does not con-
tact the fl ow-through. If residual ethanol is noticed on the out-
side of the RNeasy spin column, place the RNeasy spin column
in a new 2 ml collection tube and discard the old collection
tube. Centrifuge at full speed for 1 min.
14. (a) Place the RNeasy spin column in a new 1.5 ml collection
tube. Add 30-50

μ

l RNase-free water directly to the spin
column membrane (without touching it). Close the lid
and centrifuge for 1 min at ³8,000× g (³10,000 rpm) to
elute the RNA.
(b) Repeat this step if the RNA yield is <30

μ

g. Using only the
fi rst eluate, RNA yield will be 15-30% less than that
obtained using a second volume of RNase-free water but
the fi nal RNA concentration will be higher.
Notes: Determine the amount of tissue. Do not use more than
50 mg (100 mg for adipose tissue). Weighing tissue is the most
accurate way to quantify the tissue to use. As a rough approxi-
mation however, a 3 mm cube (volume 27 mm 3 ) of most ani-
mal tissue weighs 25-35 mg.
**Before step 1, brain tissue must be placed on dry ice through-
out. Step 1 through step 5, keep tubes at room temperature,
steps 5-9 on ice, after step 9 at room temperature. Extracted
RNA should immediately be placed on ice.

μ

Here, we describe an RNA isolation procedure that can be performed
in less than 1 h on small (<15 mg) tissue samples, using common
commercially available reagents ( 33 ):

Modifi ed RNA Isolation
Procedure (Guanidinium
Thiocyanate)

1. Homogenize fresh or frozen tissue in sterile tubes in 1 ml guani-
dinium thiocyanate (GTC) solution per 100 mg of tissue.
2. Add 1/10 vol of 2 M sodium acetate (pH 4) and vortex.
3. Add 1 vol phenol. Add 1/5 vol chloroform: isoamyl alcohol
(24:1 v / v ) and vortex.
4. Place on ice for 15 min.
5. Centrifuge at 10,000× g for 10 min.
6. Transfer upper, aqueous phase into a sterile microcentrifuge
tube. Add 95% ethanol (2:1 v / v of aqueous phase) and vortex.
7. Centrifuge at 10,000× g for 10 min. Remove supernatant.
8. Add 200 ml of 70% ethanol to RNA pellet.
9. Centrifuge at 10,000× g for 10 min. Remove supernatant.
10. Dry the RNA pellet.