Publication: Quantitative in situ hybridization for the evaluation of gene expression in asynchronous and synchronized cell cultures and in tissue sections
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Date
1999
Authors
Barlati, S. ; Zoppi, N. ; Copeta, A. ; Tavian, D. ; De Petro, G. ; Colombi, M.
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Publisher
Murcia : F. Hernández
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DOI
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info:eu-repo/semantics/article
Description
Abstract
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 SlG2 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-Hepl),
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.
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