Cellular and Molecular Biology


Quantitative in situ hybridization for the evaluation of gene expression in asynchronous and synchronized cell cultures and in tissue sections

S. Barlati, N. Zoppi, A. Copeta, D. Tavian, G. De Petro and M. Colombi

Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology,
University of Brescia, Medical Faculty, Brescia, Italy

Offprint requests to: Prof. Sergio Barlati, Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, Medical Faculty, University of Brescia, Via Valsabbina 19, 25123 Brescia, Italy. Fax: 39.030.3701157. e-mail: barlati@med.unibs.it


Summary. We describe an image analysis (IA) system that has been applied for the quantitative evaluation of mRNAs evidenced by in situ hybridization (ISH) with radiolabelled probes in cultured cells and in tissue sections. The ISH-IA method was used for the evaluation of cultured cell morphological parameters such as cell and nucleous area (CA and NA, respectively) in parallel with the levels of mRNAs detected as hybridization grains areas (GA). The evaluation of these parameters, together with the analysis of the levels of mRNAs (c-jun, cyclin A) specific for given cell cycle phases (i.e. G1 and S/G2), allowed the identification, in asynchronous cultures of human skin fibroblasts, of cells in G1 and S/G2 phases. The mRNA levels measured by ISH-AI were comparable with those detected by RT-PCR. This method was also applied for the analysis of fibronectin (FN) gene expression in control skin fibroblasts in relationship with the different phases of the cell cycle and in comparison with a tumor cell line (Sk-Hep1), heterogeneous either for morphometric parameters or for the levels of this transcript. Finally, the ISH-AI was applied for the semiquantitative evaluation of the expression, localization and alternative splicing pattern of FN mRNA in normal liver and in hepatocellular carcinoma (HCC) tissue sections. Histol. Histopathol. 14, 1231-1240 (1999)

Key words: Cell cycle, Fibronectin, Gene expression, Hepatocellular carcinoma, In situ hybridization

DOI: 10.14670/HH-14.1231