Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 168

1. Add the following components at room temperature in the order listed:

5X Buffer specifi c for RNA polymerase provided

with the polymerase by the manufacturer 4 µ L

100 m M DTT 2 µ L

Ribonuclease inhibitor (e.g., RNasin) 20-40 µ g

Stock of rATP, rGTP, and rUTP (2.5 m M each) 4 µ L

DEPC-treated dH 2 O to a fi nal volume of 20 µ L

(taking the entire reaction volume into account) X µ L

Linearized template DNA 0.2-1.0 µ g

- 32 P]rCTP (50

[ µ Ci at 10 µ Ci/ µ L, 800 Ci/mmol) 5 µ L

SP6, T3, or T7 RNA polymerase 15-20 µ g

Final volume 20 µ L

At low temperature, DNA can precipitate in the presence of the 5X buffer. It is
therefore important to keep the order of addition as listed in the protocol, and
to keep the mixture at room temperature during the addition. Specifi c activity of
[

- 32 P]rCTP can be increased to 3000Ci/mmol.

2. Incubate for 1 h at 37°C.

3. To digest the DNA template with DNase I after the transcription reaction, add
RQ1 RNase-free DNase to a concentration of 1 µ g/ µ g of template DNA and

incubate for 15 min at 37°C.

4. Separate unincorporated oligonucleotides by size-exclusion chromatography
(e.g., Nuctrap Push Columns, Stratagene). Equilibrate the column with 80 µ L 1X

TE buffer. Bring the sample volume to 80 µ L with STE buffer; load onto the

resin and elute sample with 80 µ L of STE buffer. Measure radioactivity of 1 µ L

of eluate in a scintillation counter ( see Note 10 ).

3.4.2. Labeling of cDNA Probes
The cDNA labeling is based on the method developed by Feinberg and
Vogelstein (7) , in which a mixture of random hexadeoxyribonucleotides is used
for DNA synthesis from a linear double-stranded DNA template. Kits with
all the necessary components are commercially available from a variety of
companies (e.g., Stratagene, Promega, Invitrogen, Amersham-Pharmacia). A
generic protocol is presented here ( see Note 11 ). The insert has to be cut from the
vector to ensure exclusive labeling of the DNA of interest (“template”) ( Fig. 4 ).
The DNA template needs to be separated from the vector (e.g., by separating it in
a low melting point agarose gel) and should have a concentration between 2 and

Fig. 4. (opposite page) Preparation of cDNA probes. (A) The plasmid contains
the vector backbone and the insert (“template”) with the DNA of interest. Unique
restriction sites on both sides of the insert are needed to cut out the insert. The
restriction sites can be identical, can cut in the vector backbone, but cannot cut in
the insert. (B) After the insert is cut with the appropriate restriction enzyme(s), it is
separated from the backbone (e.g., by electrophoresis). (C) The double-stranded cDNA