Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 166

Fig. 2. UV shadowing. Examine the RNA on the nylon membrane under UV light
(254 nm). After transfer, a handheld UV lamp is used to refl ect UV light onto the
membrane. The RNA can be seen as a darker band that stretches along the lanes. Two
ribosomal bands are visible on top of the mRNA. Human cerebellar RNA is shown.

8. Hold a UV lamp (254 nm) over the membrane. You should see dark bands
running down the lanes. 28S and 18S RNA bands should be visible above the
background ( Fig. 2 ). Mark 28S and 18S RNA bands with a soft pencil on the
nylon membrane. Caution: Wear proper gloves and face shield to avoid skin
exposure to UV light, and never look into the UV lamp ( see Notes 5-8 ).

J/cm 2 .
10. Store the membrane in dry place between sheets of fi lter paper.

9. UV crosslink membrane at 254 nm, 120,000 µ

3.4. Labeling of Probes
We use 32 P- or 33 P- radiolabeled DNA or RNA probes for our Northern
blots. In general, we prefer 32 P-labeled riboprobes over random-labeled DNA
probes. The advantage of riboprobes is a higher sensitivity, higher specifi c
activity (only the antisense strand is labeled, whereas both strands are labeled in
cDNA probes), lower background, and higher stringency. However, riboprobes
cannot be stripped off the blots. If blots need to be exposed to more than one
probe of similar size, the blots can be reused only once the radioactivity is
decayed. 33 P has a longer half-life than 32 P, yields crisper bands, but needs a
longer exposure time to the fi lm than 32 P. Kits for riboprobes are commercially
available (e.g., Amersham-Pharmacia, Promega, etc.) ( see Note 9 ).

3.4.1. Labeling of Riboprobes

Transcription of RNA is performed with the appropriate RNA polymerase
(T3, T7, or SP6), depending on the RNA polymerase promoter sites present in
the vector used. The plasmid is linearized at an appropriate restriction site at
the opposite end of the antisense polymerase site, and “run-off” transcripts
are obtained, which should not contain vector sequences ( Fig. 3 ). Antisense
RNAs are needed for the positive reactions, while sense RNAs are used as
negative controls.