Transcript Diapositive 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 Génomique Structurales de l’IGBMC, UPR 9004 du CNRS, 1 rue Laurent Fries, 67404 Illkirch
&Laboratoire de Biologie Moléculaire, d'Analyse Génique et de Modélisation CRP-Santé 42, rue du Laboratoire 1011 Luxembourg
Web site: www.microarray.lu
Introduction
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.
Objectives
Cytoskeleton changes during
epithelial-mesenchymal transition
To develop a function-focused oligonucleotide microarray dedicated to the transcriptome analysis of actin cytoskeleton genes.
Experimental procedure
• The sequences of genes related to the actin cytoskeleton functions were collected and stored in a database named Actinome.
• A bioinformatic program for the design of oligonucleotide microarray probe was developed and validated.
• 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 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.
Results
Implementation
procedure of ActiChip
1
Actinome : Actin cytoskeleton integrative genomic database
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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.
Sequence
Retrieval
AAAAA
AAAAA
AAAAA
Actin mRNA
(% identity)
α-actin skeletal
muscle
actin aortic
smooth
muscle
α-actin
cardiac
muscle
α-actin skeletal muscle
100 %
85,4 %
85,4 %
β-actin
γ-actin
cytoplasmic
γ-actin enteric
smooth muscle
86,4 %
86,6 %
84,8 %
2
Actinome includes genomic data ( 1 genome localisation, 2 exonic map), and sequence analysis features ( 3 GO,
multiple alignment, protein family clustering, tissue expression).
Design of oligonucleotide sequences for ActiChip
Actinome
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.
Probe Design
1
A home-made program was used to generate 60-mer probe sequences from Actinome.
The program allows the user to select fitting parameters (length, GC content, Tm melting temperature, specificity…) and offers an interactive
graphical interface ( 1 ) displaying the complete set of sequences being selected in a highly comprehensive way.
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).
We automatically design a unique probe for 75 % of the database, and the 25 % are refined manually (Tm adjustment, distance from 3’ end …).
Printing of a Prototype ActiChip containing 59 oligonucleotides
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.
ActiChip prototype
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 µM in 1x microspotting plus buffer (Arrayit).
Validation of probe design with cells harbouring well-characterised expression profiles
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.
ActiChip performances:
Skeletal muscle
(Cy5)
Human mammary carcinoma
(Cy3)
•Homogenous and regular size spots,
•Good dynamic range,
•Low background noise,
•Good signal-to-noise ratio.
Control genes
Cy5
Cy3
Obs./ Exp. Obs./ Exp.
α-actin skeletal muscle
γ-actin enteric smooth muscle
α-actin cardiac muscle
γ-actin cytoplasmic
actin aortic smooth muscle
β-actin
E-cadherin
Zyxin
Tropomyosin 1 α
Cytokeratin 1
Cytokeratin 8
Cytokeratin 18
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93 % of detected genes correlate with expected pattern,
Conclusions and perspectives
• 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 implementation of a bioinformatic tool for actin cytoskeleton probe design.
• The designed probes specifically react with genes harbouring high % identities (e.g. actins).
4 % were false positive and need further refinement.
• 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.
Acknowledgments: Ministère de la Culture, de l’Enseignement Supérieur et de la Recherche du Luxembourg, Fonds National de la recherche du Luxembourg,CNRS, France