1. ActiChip, a dedicated microarray for gene expression profiling of actin cytoskeleton associated genes Jean Muller*&, Laurent Vallar&, Guillaume Vetter&, Evelyne Friederich& and Olivier Poch* *Laboratoire de Biologie et de G é nomique Structurales de l ’ IGBMC, UPR 9004 du CNRS, 1 rue Laurent Fries, 67404 Illkirch &Laboratoire de Biologie Mol é culaire, d'Analyse G é nique et de Mod é lisation CRP-Sant é 42, rue du Laboratoire 1011 Luxembourg Web site: www.microarray.lu Results The actin cytoskeleton is a dynamic meshwork of proteaneous filaments, with an intrinsic capability to rapidly assemble and disassemble, involved in cell shape regulation, cell motility and adhesion (lamellipodium, filopodia). The actin genes also encode a family of proteins that are highly conserved through evolution of eukaryotes. In cancer cells, structural and functional perturbations of the actin cytoskeleton correlate with higher proliferation rates and uncontrolled movement. A modification of the expression pattern of actin-associated proteins is observed during this process making the actin cytoskeleton an interesting target for the development of novel cancer therapies and diagnostic tools ( Giganti, A., Progress in cell cycle research, 2003 ). Microarray-based high throughput analysis of gene expression networks (transcriptomic analysis) allows to gain an integrated view on complex biological processes such as cytoskeletal alterations. These studies rely on bioinformatic exploration tools of biological databases in the context of high throughput biology. Introduction Experimental procedure Skeletal muscle (Cy5) Human mammary carcinoma (Cy3) AAAAA Actinome Probe Design ActiChip prototype Sequence Retrieval Implementation procedure of ActiChip A prototype of the ActiChip was manufactured based on a limited series of probes and was assessed through the transcriptome analysis of cells with well-characterised gene expression profiles. The sequences of genes related to the actin cytoskeleton functions were collected and stored in a database named Actinome. A collection of long oligonucleotide was implemented from Actinome with the aim at developing a microarray dedicated to the expression profile of human genes related to the actin cytoskeleton (ActiChip). A bioinformatic program for the design of oligonucleotide microarray probe was developed and validated. Objectives To develop a function-focused oligonucleotide microarray dedicated to the transcriptome analysis of actin cytoskeleton genes. Printing of a Prototype ActiChip containing 59 oligonucleotides Validation of probe design with cells harbouring well-characterised expression profiles 93 % of detected genes correlate with expected pattern, 4 % were false positive and need further refinement. ActiChip performances: Homogenous and regular size spots, Low background noise, Good signal-to-noise ratio. Good dynamic range, 59 marker genes were selected from the probe collection based on their characteristic tissue expression profiles in epithelium/mesenchyme and/or in muscle. Several positive (ubiquitous genes HSP70, β-actin) and negative (poly (A), E. coli) control probes, as well as a quality and normalisation probe set (A. thaliana spike RNA, Wang and al Genome Biology 2003) were also included in the probe list. HPLC-purified synthetic oligonucleotides were spotted in quadruplicate using split pins in a Microgrid II microarrayer (Biorobotics, USA) on pre-treated glass slide (SuperEpoxy, Arrayit, USA) allowing their covalent attachment to the surface. Spotting was carried out in a 50 % humidity controlled atmosphere at 20 °C. Probe concentration was adjusted to 40 µ M in 1x microspotting plus buffer (Arrayit). Acknowledgments: Minist è re de la Culture, de l ’ Enseignement Sup é rieur et de la Recherche du Luxembourg, Fonds National de la recherche du Luxembourg,CNRS, France Poly(A)+RNA were purified from human mammary carcinoma cell (MCF-7) and from human skeletal muscle (Ambion), and were indirectly labelled with cyanine 3 and cyanine 5, respectively. Equimolar target mix were hybridised to the ActiChip prototype at 42°C for 18h. The hybridised slides were scanned using a 4000B microarray reader (Axon, USA), and fluorescence signal intensities were determined using the Genepix Pro 4.1 software. Conclusions and perspectives α-actin skeletal muscle Tropomyosin 1 α actin aortic smooth muscle Cytokeratin 18 β-actin E-cadherin Zyxin γ-actin cytoplasmic γ-actin enteric smooth muscle α-actin cardiac muscle Cytokeratin 1 Cytokeratin 8 + + + - + + + + - - - + + + + - + + + + - - - + - - - + + + + + - - - + - - - + + + + + - - - + Control genesCy5Cy3 Obs./ Exp. Actinome : Actin cytoskeleton integrative genomic database Design of oligonucleotide sequences for ActiChip Gene accession numbers were retrieved from GenBank based on bibliography and Gene Ontology searches. The initial set of sequences included 1164 entries related to actin cytoskeleton functions (structural and signalling components). Many of them were highly similar sequences (ex: actin, myosin, tropomyosin, cytokeratin), splice variants and pseudogenes making the design of specific oligonucleotide probes complex. A home-made program was used to generate 60-mer probe sequences from Actinome. Actinome includes genomic data ( genome localisation, exonic map), and sequence analysis features ( GO, multiple alignment, protein family clustering, tissue expression). The selected probes are validated by crossed-selection between standard human databases, Unigene (Wheeler, D.L., NAR, 2003) and RefSeq (Pruitt, K.D., NAR, 2003). Long oligonucleotide probes (>50mer) have several main advantages when compared to cDNA: their hybridisation properties can be chosen so that they are homogeneous, the detection is more specific and equivalent in sensitivity. 1 The implementation of a bioinformatic tool for actin cytoskeleton probe design. The development of Actinome database a useful tool dedicated to the transcriptome analysis of actin cytoskeleton-related genes. The database will be permanently updated with new entries (newly identified genes/proteins). The designed probes specifically react with genes harbouring high % identities (e.g. actins). In future: ActiChip with >1000 probes for gene expression profiling experiments. Experimental data will be compared to those obtained with the same tissue samples using Affymetrix and cDNA array results. 1 Actin mRNA (% identity) α-actin skeletal muscle actin aortic smooth muscle α-actin cardiac muscle β-actin γ-actin cytoplasmic γ-actin enteric smooth muscle α-actin skeletal muscle100 %85,4 % 86,4 %86,6 %84,8 % The program allows the user to select fitting parameters (length, GC content, T m melting temperature, specificity…) and offers an interactive graphical interface ( ) displaying the complete set of sequences being selected in a highly comprehensive way. We automatically design a unique probe for 75 % of the database, and the 25 % are refined manually (T m adjustment, distance from 3 ’ end … ). 3 2 132 1 Cytoskeleton changes during epithelial-mesenchymal transition