Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 145

(c) 10× Loading dye:
0.25% Bromophenol blue.
0.25% Xylene cyanol.
20% Ficoll.
Dissolve in DEPC water. Alternately, any commercially avail-
able RNase-free loading dye may be used, provided the
migration rate is known.
(d) 20× SSPE:
175.3 g of NaCl.
27.6 g of NaH 2 PO 4 × H 2 O.
7.4 g of EDTA.
Adjust pH to 7.4 in a 700 ml volume then and bring to fi nal
volume of 1 L.

1. Denaturing agarose-formaldehyde gel electrophoresis of RNA:
(a) Prepare a 1% agarose gel. For 250 ml, dissolve 2.5 g aga-
rose with 25 ml of 10× MOPS and 205 ml of water. When
the fl ask has cooled, add 12 ml formaldehyde. Pour the gel
and allow it to set at room temperature. Place the gel in a
gel tank and add 1× MOPS running buffer. Note:
Formaldehyde is toxic and casting should be carried out in
a fume hood and the gel should be covered when in use.
Cast the gel as thin as practical as this will enhance transfer
of high molecular weight RNA to the membrane.
(b) Load samples onto the gel. During the fi nal step of RNA
extraction samples should be resuspended in at least 12

3.3.2. Procedures

l
of sample buffer. Heat samples for 5 min at 65°C, place on
ice, and then add 3

μ

l of loading buffer.
(c) Run the gel at 100-200 V. The length of time will vary
depending on the length of your gel, but a general rule is
to run until the bromophenol blue has migrated two-
thirds of the length. Image the gel with a UV transillumi-
nator and document in the presence of a fl uorescent ruler.
Place gel in 20× SSPE while preparing the next step.
2. Capillary transfer of RNA to a membrane support:
(a) Fill the reservoirs of an electrophoresis tank with 20×
SSPE. Cut a wick from a piece of Whatman 3MM paper
long enough to span both reservoirs. Cut four pieces of
Whatman 3MM paper the size of the gel, wet with 20×
SSPE, and place two in the center of the tank on top of the
wick. Place the gel on the fi lter paper and roll out air bub-
bles. Cut four strips of parafi lm and place under each edge
of the gel. Cut a piece of positively charged nylon mem-
brane (Ambion) the dimensions of the gel and position on

μ