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Quantification of the Transcripts Encoding Different Forms of AChE in Various Cell Types: Real-Time PCR Coupled with Standards in Revealing the Copy Number

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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 × 104 in PC12 (rat neuronal cell), 1.3 × 104 in Caco-2 (human intestinal cell), 0.67 × 104 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 × 104, 3.3 × 104, 2.7 × 104, 133.3, and 46.7, respectively, and AChER were 0.07 × 104, 0.13 × 104, 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.

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Abbreviations

AChE:

Acetylcholinesterase

GPI:

Glycophosphatidylinositol

NGF:

Nerve growth factor

EPO:

Erythropoietin

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Acknowledgments

This research was supported by Hong Kong Research Grants Council Theme-based Research Scheme (T13-607/12R), GRF (S-HK015/09, 662911, 663012, 662713, N_HKUST629/13), The Hong Kong Jockey Club Charities Trust and Foundation of The Awareness of Nature (TAON12SC01) to Karl Tsim.

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Authors and Affiliations

  1. Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China

    Cathy W.C. Bi, Wilson K.W. Luk, Yuen H. Liu, Li Xu, Kei M. Lau, Miranda L. Xu, Roy C.Y. Choi & Karl W.K. Tsim

  2. Instituto de Neurociencias de Alicante, Universidad Miguel Hernández-CSIC, Sant Joan d’Alacant, 03550, Spain

    María-Letizia Campanari & Javier Sáez-Valero

  3. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), San Juan de Alicante, Spain

    María-Letizia Campanari & Javier Sáez-Valero

Authors
  1. Cathy W.C. Bi
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  2. Wilson K.W. Luk
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  3. María-Letizia Campanari
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  4. Yuen H. Liu
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  5. Li Xu
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  6. Kei M. Lau
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  7. Miranda L. Xu
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  8. Roy C.Y. Choi
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  9. Javier Sáez-Valero
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  10. Karl W.K. Tsim
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Corresponding author

Correspondence to Karl W.K. Tsim.

Additional information

Cathy W.C. Bi and Wilson K.W. Luk contributed equally to this study.

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Supplementary Figure

Standard curves of different rat and human AChE plasmids. Different plasmids of rat and human AChE splicing variants were applied to the standard protocol in Applied Biosystems 7500 Fast to generate the standard curves. Threshold values represented a certain florescent intensity exhibited by SYBR Green Fluorescent Stain actively binding onto double-strand DNA. The plasmid standard constructed were subjected to sequential 10 fold dilution, giving a rage of 102 to 109 numbers of copies of plasmid standard. These dilutions were subjected for real-time PCR amplification. Data are expressed as ΔΔCt value. Mean ± SD, n = 3. (JPEG 75 kb)

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Bi, C.W., Luk, W.K., Campanari, ML. et al. Quantification of the Transcripts Encoding Different Forms of AChE in Various Cell Types: Real-Time PCR Coupled with Standards in Revealing the Copy Number. J Mol Neurosci 53, 461–468 (2014). https://doi.org/10.1007/s12031-013-0210-6

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  • DOI: https://doi.org/10.1007/s12031-013-0210-6

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