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Isolation and analysis of genes mainly expressed in adult mouse heart using subtractive hybridization cDNA library

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Abstract

Subtractive hybridization cDNA library (SHL) is one of the powerful approaches for isolating differentially expressed genes. Using this technique between mouse heart and skeletal muscle (skm) tissues, we aimed to construct a cDNA-library that was specific to heart tissue and to identify the potential candidate genes that might be responsible for the development of cardiac diseases or related pathophysiological conditions. In the first step of the study, we created a cDNA-library between mouse heart and skm tissues. The homologies of the randomly selected 215 clones were analyzed and then classified by function. A total of 146 genes were analyzed for their expression profiles in the heart and skm tissues in published mouse microarray dataset. In the second step, we analyzed the expression patterns of the selected genes by Northern blot and RNA in situ hybridization (RISH). In Northern blot analyses, the expression levels of Myl3, Myl2, Mfn2, Dcn, Pdlim4, mt-Co3, mt-Co1, Atpase6 and Tsc22d1 genes were higher in heart than skm. For first time with this study, expression patterns of Pdlim4 and Tsc22d1 genes in mouse heart and skm were shown by RISH. In the last step, 43 genes in this library were identified to have relationships mostly with cardiac diseases and/or related phenotypes. This is the first study reporting differentially expressed genes in healthy mouse heart using SHL technique. This study confirms our hypothesis that tissue-specific genes are most likely to have a disease association, if they possess mutations.

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Acknowledgments

We thank Sema Bilgic (PhD) and Mehves Poda (PhD) for their contributions in the application of RISH technique also Neslihan Coban (PhD student) for her contributions in selections of positive clones from cDNA library and isolations of plasmid DNA. This study was supported by State Planning Organization of Turkey and Scientific Research Projects Coordination Unit of Istanbul University (Project numbers: T–1062/19022001, T–901/02062006, BYP-3135, ACIP-3107).

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  1. Department of Genetics, Institute for Experimental Medicine, Istanbul University, Vakif Gureba Cad., 34080, Sehremini, Istanbul, Turkey

    Evrim Komurcu-Bayrak, Bilge Ozsait & Nihan Erginel-Unaltuna

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  1. Evrim Komurcu-Bayrak
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  2. Bilge Ozsait
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Correspondence to Nihan Erginel-Unaltuna.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11033_2012_1653_MOESM1_ESM.doc

Supplementary Table 1 (Online Resource 1). The determinations of differential expressed genes in heart according to skeletal muscle using published mouse microarray dataset in GEO database. Supplementary material 1 (DOC 355 kb)

11033_2012_1653_MOESM2_ESM.doc

Supplementary Table 2 (Online Resource 2). The detailed information of the probes in Northern Blot and RISH techniques. Supplementary material 2 (DOC 39 kb)

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Komurcu-Bayrak, E., Ozsait, B. & Erginel-Unaltuna, N. Isolation and analysis of genes mainly expressed in adult mouse heart using subtractive hybridization cDNA library. Mol Biol Rep 39, 8065–8074 (2012). https://doi.org/10.1007/s11033-012-1653-5

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