Biomedical Engineering Reference
In-Depth Information
11
Quantifi cation of mRNA in Neuronal Tissue
by Northern Analysis
Christine L. Konradi
1. Introduction
RNA is transcribed in the cell nucleus from a DNA template with ribo-
nucleotides as the building blocks. As RNA is transported out of the nucleus,
it is spliced; that is, predetermined sequences (introns) are cut out of the
transcript. If certain introns serve as either exons or introns, “alternative
splicing” can occur and one gene can give rise to several different size RNA
transcripts. The spliced RNA, the mRNA, is released into the cytosol and
translated into protein.
Levels of particular mRNA transcripts are assayed in Northern blots. For
Northern blots, tissue is homogenized and RNA transcripts are separated
from DNA, proteins, and other contaminants. The purifi ed RNA is then size
separated on a denaturing agarose gel by electrophoresis. RNA is transferred
to a nylon membrane, which is reacted with a known, labeled, antisense
RNA or DNA molecule (“probe”). This molecule will fi nd its counterpart on
the membrane and bind to it. The labeled probe is then visualized. Because the
probe is presented in excess, the amount of labeling is a representation of
the amount of RNA in the original sample. The most common way of labeling the
probe is by incorporating radioactive nucleotides. Luminescence can be used
as well, but it is less sensitive. Either way, X-ray fi lm is used to visualize
the probe location and probe quantity on the membrane. Alternatively, image
analysis systems are used instead of X-ray fi lms.
RNA is degraded by RNases. RNases are found within the same tissues
as RNA. It is therefore important to separate RNases from RNA quickly and
Search WWH ::
Custom Search