title: Quantification of the transcripts encoding different forms of AChE in various cell types: real-time PCR coupled with standards in revealing the copy number.
abstract: Acetylcholinesterase (AChE) is encoded by a single gene, and the alternative splicing at the 3' end produces different isoforms, including tailed (AChET), read-through (AChER), and hydrophobic (AChEH). Different forms of this enzyme exist in different cell types. Each AChE form has been proposed to have unique function, and all of them could be found in same cell type. Thus, the splicing process of different AChE forms remains unclear. Here, we aimed to establish a quantification method in measuring the absolute amount of each AChE splicing variants within a cell type. By using real-time PCR coupled with standard curves of defined copy of AChE variants, the copies of AChET transcript per 100 ng of total RNA were 5.7 × 10(4) in PC12 (rat neuronal cell), 1.3 × 10(4) in Caco-2 (human intestinal cell), 0.67 × 10(4) in TF-1 (human erythropoietic precursor), 133.3 in SH-SY5Y (human neuronal cell), and 56.7 in human umbilical vein endothelial cells (human endothelial cells). The copies of AChEH in these cell types were 0.3 × 10(4), 3.3 × 10(4), 2.7 × 10(4), 133.3, and 46.7, respectively, and AChER were 0.07 × 10(4), 0.13 × 10(4), 890, 3.3, and 2.7, respectively. Furthermore, PC12 and TF-1 cells were chosen for the analysis of AChE splicing pattern during differentiation. The results demonstrated a selective increase in AChET mRNA but not AChER or AChEH mRNAs in PC12 upon nerve growth factor-induced neuronal differentiation. PC12 cells could therefore act as a good cell model for the study on alternative splicing mechanism and regulation of AChET.
authors.affiliation: Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China.
authors.forename: Cathy W C
authors.initials: CW
authors.lastname: Bi
authors.forename: Wilson K W
authors.initials: WK
authors.lastname: Luk
authors.forename: María-Letizia
authors.initials: ML
authors.lastname: Campanari
authors.forename: Yuen H
authors.initials: YH
authors.lastname: Liu
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.forename: Kei M
authors.initials: KM
authors.lastname: Lau
authors.forename: Miranda L
authors.initials: ML
authors.lastname: Xu
authors.forename: Roy C Y
authors.initials: RC
authors.lastname: Choi
authors.forename: Javier
authors.initials: J
authors.lastname: Sáez-Valero
authors.forename: Karl W K
authors.initials: KW
authors.lastname: Tsim
mesh_terms: D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D018938:Caco-2 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009928:Organ Specificity; D016716:PC12 Cells; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D007527:Isoenzymes; D012333:RNA, Messenger; D000110:Acetylcholinesterase