Biology Reference
In-Depth Information
demonstrated to exert significant effects upon both early and long-term gene
transcription, thereby mimicking the more classically “transcriptionally ori-
ented” growth factor receptor systems. The presence of well-established
transcriptomic analytical technologies therefore allows for the facile analysis
of GPCR-mediated transcriptional signatures. As we have previously dis-
cussed, it appears that the
b
-arrestin-mediated GPCR signaling paradigm
represents one of the primary GPCR-controlled transcriptional regulatory
mechanisms.
26
While gene array technologies were originally developed
to create profiles of distinctly regulated transcripts, often created through
long-term experimental paradigms,
88,89
they are also highly suited to inves-
tigation of both short- and long-term effects of receptor stimulation.
26,60
For
a simple analysis of transcriptional activity induced by receptor stimulation,
multiple (at least in triplicate) control and test condition samples are analyzed
in a direct comparative manner. Upon the creation of the first series of
reproducible arrays, methods such as primary data selection, statistical anal-
ysis, and quality control processes were applied to the datasets generated.
90
Many of first transcriptomic array technologies employed radioactive detec-
tion methods, but due to vagaries of spot detection and reproducibility, these
were quickly superseded by differential fluorescent platforms. Employing a
differential-sample fluorescent dye labeling process (Cy3/Cy5), quantitative
changes in mRNA expression are now easily obtainable on both a large
(
>
22,000 genes) or small (100 genes) scale.
91,92
As with most technologies
based upon fluorescent labeling, one of the most problematic issues before
data can be extracted is the inability to completely separate unbound dye
from that associated with the sample. Subtraction of such background inten-
sity is achieved by statistically computing the average background intensity
and using the standard deviation among this intensity to calculate a confi-
dence interval, the upper limit of which is used for the subsequent back-
ground correction. As array samples should be assessed with high
numbers of replicates, it may be necessary to assess GPCR transcriptomic
activity across multiple array chips. The comparison of multiple gene regu-
lation profiles between microarray chips therefore requires the application of
efficient dataset normalization. Current normalization processes, however,
often rely upon the use of “housekeeping” genes, whose expression profiles
are assumed to be largely resistant to experimental variation. However, with
respect to our current thinking of physiological response/signal transduction
networks, the concept of a truly nonchanging transcript on the array unfor-
tunately becomes less and less likely.
20
Internal spotted standards of a control
transcript, for example, serum albumin, can provide an adequate control for
Search WWH ::
Custom Search