Nucleic Acids Research, Vol 27, Issue 10 2211-2218, Copyright © 1999 by Oxford University Press
H von der Kammer, C Albrecht, M Mayhaus, B Hoffmann, G Stanke and RM Nitsch
In order to identify genes that are regulated by muscarinic acetylcholine
receptors, we developed an mRNA differential display technique (DD)
approach. By increasing redundancy and by evaluating optimised reagents and
conditions for reverse transcription of total RNA, PCR and separation of
PCR products, we generated a DD protocol that yields highly consistent
results. A set of 64 distinct random primers was specifically designed in
order to approach a statistically comprehensive analysis of all mRNA
species in a defined cell population. This modified DD protocol was applied
to total RNA of HEK293 cells stably expressing muscarinic m1 acetylcholine
receptors and cells stimulated with the receptor agonist carbachol were
compared to identical but non-stimulated cells. In 81 of 192 possible PCR
experiments, 38 differential bands were identified. Sequence analysis
followed by northern blot analyses confirmed differentially expressed genes
in 19 of 23 bands analysed. These represented 10 distinct immediate-early
genes that were up-regulated by m1AChR activation: Egr- 1, Egr-2, Egr-3,
NGFi-B, ETR101, c- jun, jun -D, Gos-3 and hcyr61, as well as the unknown
gene Gig-2. These data show that this improved DD protocol can be readily
applied to reliably identify differentially expressed genes.
ARTICLES
Identification of genes regulated by muscarinic acetylcholine receptors: application of an improved and statistically comprehensive mRNA differential display technique
Center for Molecular Neurobiology Hamburg, University of Hamburg, Martinistrasse 52, D-20246 Hamburg, Germany. kammer@plexus.uke.uni- hamburg.de
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