Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 144

3.3. Northen Blotting

Though it has been superseded in some aspects by other RNA
analysis techniques discussed in this chapter, Northern analysis
remains a powerful method for quantifi cation and characterization
of transcription. We may still apply Northern analysis to obtain a
relative comparison of abundance between samples on a single sup-
port and it remains the simplest method for determination of tran-
script size and hence, detection of alternate splicing.
One of the strengths in performing a Northern blot is in its
relative simplicity and low cost compared to other RNA character-
ization methods, making it accessible to most laboratories ( 49, 50 ).
Purifi ed RNA samples are separated by size via denaturing gel
electrophoresis, and then the RNA is transferred to a membrane,
cross-linked, and probed for the gene of interest with a labeled
nucleic acid. The procedure is fl exible and customizable to your
particular laboratory (e.g., Probes may be anything from p32/p33
radiolabeled DNA, non-radiolabeled DNA, RNA, or oligonucle-
otides may all be used).
As with other RNA characterization methods, selecting the
correct assay for your experimental paradigm requires consider-
ation of the limitations of each system. Northern blots are particu-
larly sensitive to RNA degradation ( 51 ). If the integrity of the RNA
is not maintained (Clear 28S Ribosomal bands of double intensity
of the 18S), then the signal from many probes may be reduced or
lost. As with all the techniques throughout this chapter, RNase-
free reagents and techniques are required for each step. Additionally,
Northern blots generally require substantial amounts of RNA
(5-50 g or greater); quantities not always generated depending

on the brain region or blood sample being examined. Lower quan-
tities of starting RNA generally yield less sensitive results depend-
ing on the abundance of the transcript being probed, but in almost
all cases Northerns will be less sensitive than qRT-PCR ( 52 ).

μ

(a) 10× MOPS solution:
50 mM anhydrous sodium acetate.
10 mM EDTA.
0.2 M MOPS.
Bring volume to 700 ml with DEPC-treated water and adjust
the pH to 6.5. Bring to fi nal volume of 1 L.
(b) RNA sample buffer (1 ml):
500

3.3.1. Materials
and Reagents

μ l deionized formamide.

180 μ l formaldehyde (37%).

216 μ l DEPC water.

100 μ l of 10× MOPS.

4 μ l EtBr (from stock of 10 mg/ml).