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

In order to identify genes that are regulated by muscarinic acetylcholine r eceptors, we developed an mRNA differential display technique (DD) approach . By increasing redundancy and by evaluating optimised reagents and conditi ons for reverse transcription of total RNA, PCR and separation of PCR produ cts, we generated a DD protocol that yields highly consistent results. A se t of 64 distinct random primers was specifically designed in order to appro ach a statistically comprehensive analysis of all mRNA species in a defined cell population, This modified DD protocol was applied to total RNA of HEK 293 cells stably expressing muscarinic mi 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 differe ntial bands were identified. Sequence analysis followed by northern blot an alyses 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, Egv-3, NGFi-B, ETR101, c-jun, jun-D, G os-3 and hcyr61, as well as the unknown gene Gig-2. These data show that th is improved DD protocol can be readily applied to reliably identify differe ntially expressed genes.