Cell and tissue microarray technologies for protein and nucleic acid expression profiling

Abstract

Tissue microarray (TMA) and cell microarray (CMA) are two powerful techniques that allow for the immunophenotypical characterization of hundreds of samples simultaneously. In particular, the CMA approach is particularly useful for immunophenotyping new stem cell lines (e.g., cardiac, neural, mesenchymal) using conventional markers, as well as for testing the specificity and the efficacy of newly developed antibodies. We propose the use of a tissue arrayer not only to perform protein expression profiling by immunohistochemistry but also to carry out molecular genetics studies. In fact, starting with several tissues or cell lines, it is possible to obtain the complete signature of each sample, describing the protein, mRNA and microRNA expression, and DNA mutations, or eventually to analyze the epigenetic processes that control protein regulation. Here we show the results obtained using the Galileo CK4500 TMA platform.

Figure 1.

Immunocharacterization of six cell lines by cell microarray technology. After cell collection, pellets deriving from six different cell lines were resuspended in agarose and then embedded in paraffin. Two consecutive sections were analyzed for SEL1L expression by immunohistochemistry (A, C) and immunofluorescence (B, D) techniques (using the monoclonal antibody described by Orlandi et al. 2003), showing the typical perinuclear/cytoplasmic subcellular location of this protein. In the immunohistochemical images, the brown color reflects SEL1L expression and nuclei were counterstained with hematoxylin; differently, in immunofluorescence analysis, SEL1L is represented in green and nuclei in blue (counterstaining with Hoechst 33258, HO). Scale bars = 10 µm.

Figure 2.

Immunophenotypical analysis of tissue microarray (TMA) slices to identify nuclear, cytoplasmic, and membrane antigens. Five different tumoral and three normal tissues were used to construct a TMA, from which three sequential slices were analyzed for pH3 (A) and SEL1L (B; monoclonal antibody described by Orlandi et al. 2002) expression by immunohistochemistry and concurrently for SEL1L and E-cadherin (C) by immunofluorescence. The immunohistochemical figures show SEL1L and H3 proteins in brown, whereas nuclei were counterstained with hematoxylin. In the immunofluorescence study, SEL1L is depicted in green, E-cadherin in red, and nuclei in blue (counterstaining with Hoechst 33258, HO). Scale bars = 25 µm.

Figure 3.

Nucleic acids extraction from tissues cored by the Galileo CK4500 Arrayer. Tissue cores were processed to extract both DNA (A) and RNA, showing good yields, but with an expected variability depending on the tissue quality and cellular content (D). The DNA can be used to amplify genes of interest (B), whereas the total RNA is suitable for RT-PCR (C) or to analyze the expression of specific microRNAs (E).