Identification of genes regulated by muscarinic acetylcholine receptors: application of an improved and statistically comprehensive mRNA differential display technique.

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.