Biochemical and Biophysical Research Communications
Identification and distribution of different mRNA variants produced by differential splicing in the human phosphodiesterase 9A gene
Section snippets
Materials and methods
Cell culture. HeLa cells (human cervix carcinoma cell line) were grown at 37 °C under 5% CO2 in Dulbecco’s modified Eagle’s medium with 10% foetal bovine serum. Jurkat cells (human T cell leukemia cell line) were cultured, harvested, and resuspended as previously described [13]. Caco2 cells (human colon adenocarcinoma cell line) were grown at 37 °C under 5% CO2 in Dulbecco’s modified Eagle’s medium with 10% foetal bovine serum, 2 mM l-glutamine, and 1% non-essential amino acids.
RNA blot and dot
Accumulation of PDE9A mRNA: RNA and dot blot analyses
In order to find an appropriate system to study the accumulation of PDE9A mRNA and its population of splice variants, total RNA of HeLa and Jurkat cells was isolated. An RNA blot was then hybridised with a 32P-radiolabelled oligonucleotide (nt 541–587) probe. A band was found around 1.9 kb (Fig. 1), in accordance with the expected size of the PDE9A mRNA. The band often appears broad or as a smear, which may indicate the existence of several mRNAs for the same gene.
The accumulation pattern of the
Acknowledgements
We thank David Piñeyro and Dr. Mònica Torras for continuous interest and help. We also thank Dr. Arsenio Nueda from Laboratorios Almirall-Prodesfarma S.A. (Barcelona) and Dr. Ferran Azorı́n (IBMB-CSIC, Barcelona) for providing HeLa, Jurkat, and Caco2 cells. This work was supported by a grant from the Spanish Ministerio de Ciencia y Tecnologı́a, (Proyectos FEDER) and it has been carried out within the framework of the Centre de Referència de Biotecnologia de la Generalitat de Catalunya.
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