Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 146

the surface of the gel. Place the remaining two pieces of
Whatman 3MM paper on top of the membrane, roll out
any air bubbles. Stack paper towels on top of the Whatman
3MM paper to a height of approximately 10 cm, and add
a light weight. Leave to transfer overnight. Faster com-
mercially available transfer methods, such as vacuum and
electroblotting can also be used.
(b) Rinse the membrane with 5× SSPE and then allow to dry.
Place in a UV cross-linker to bind the RNA to the mem-
brane. Alternately, the membrane may be baked at 80°C
for 2 h.
3. Hybridization of labeled probes:
(a) Prepare labeled probes specifi c to your requirements (e.g.,
Prime-It ® II Random Primer Labeling Kit, Stratagene).
Prehybridize the membrane in 5 ml of hybridization solu-
tion (5× SSPE, 50% ( w / v ), 5× Denhardt's solution, 1% SDS,
10% Dextran Sulfate Na salt). Incubate with rotation for
1-3 h at 42°C for DNA probes (60°C for RNA probes).
(b) Denature probes by heating for 5 min at 95°C and transfer
briefl y to ice; then, add the probe to the hybridization mix
and incubate with rotation overnight.
(c) Wash the membrane twice for 5 min with 2× SSPE at room
temperature, and two 5 min incubations at 65°C with 2×
SSPE, 2% SDS. If signal is still strong, additional washes
with 0.1× SSPE, 1% SDS may be needed. Rinse membrane
in 5× SSPE, cover in plastic wrap, and expose to Kodak
XAR fi lm in a cassette containing X-ray intensifying screens
at −80°C for overnight up to 1 week.

3.4. Reverse
Transcription
Polymerase Chain
Reaction

Reverse transcription PCR (RT-PCR) is the most sensitive tech-
nique for mRNA detection and quantitation currently available
( 53 ). This method permits the analysis of incredibly small quanti-
ties of nucleic acid, as little as one cell equivalent, which is not
possible with traditional methods. cDNA microarrays can contain
large percentages of improperly annotated probes which can cre-
ate false-positive gene hits. In addition, nonspecifi c hybridization
or cross-hybridization of closely related genes to oligo or cDNA
probes can also yield false positives. Thus, RT-PCR has become
the preferred method for validating results obtained from array
analyses and other techniques that evaluate gene expression on a
global scale.
RT-PCR is a variant of the PCR, where an RNA strand is fi rst
reverse transcribed into its DNA complement (complementary
DNA, or cDNA) using the enzyme reverse transcriptase, and the