Microscope glass slides (76 × 26 × 1 mm3, Knittel, Merck ABS, Postfach, Germany) or glass cover slips (4 mm diameter, 0.15 mm thick, PolyLabo, Strasbourg, France) were obtained from commercial sources. All solvents were purchased from Fluka Chemicals (Buchs, Switzerland) unless otherwise specified. Buffers were prepared with deionised water and micro-filtered (0.22 µ, Millipore GmbH, Eschborn, Germany). Oligonucleotides were obtained from Eurogentec S.A. (Brussels, Belgium). Water soluble heterobifunctional cross-linkers, sulfonated analogues (s-), s-MBS (m-maleimidobenzoyl-N-hydroxysulfo-succinimide ester), s-SIAB [sulfosuccinimidyl(4-iodoacetatyl)aminobenzoate], s-SMCC [sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1 carboxylate], s-GMBS [N-(γ-maleimidobutyryloxy)sulfo succinimide ester], s-SMPB [sulfosuccinimidyl 4-(p-maleimidophenyl)-butyrate] and the corresponding non-water soluble forms (MBS, SIAB, SMCC, GMBS) were purchased from Pierce Chemicals (Rockford, IL). Fluorescent measurements were performed using an inverted epi-fluorescence microscope equipped with an arc mercury lamp (Axiovert S100TV + HBO 100W/2, Carl Zeiss, Oberkochen, Germany) and coupled to a CCD camera (Princeton Instrument Inc., Trenton, NJ). Fluorescent signals were measured by integration of the signal in an image with in-house developed software. HPLC analyses were run on a HP1090 Liquid Chromatography (Hewlett-Packard, Agilent Technologies, Palo Alto, CA) using an Aquapore C18 column (RP300, 7 µm particle size, ABI, Foster City, CA). Phosphorimaging was performed on the Molecular Imager FX (BioRad, Richmond, CA) and radioactive signals analysed with the Quantity One Biorad software.
Nucleic acid preparation: (i) synthetic oligonucleotides
Oligonucleotides were designed as follows;
The 10 Ts serve as a link to enhance hybridisation efficiency as previously shown (14
). The 5′-ends of oligonucleotides up1
were modified during synthesis with one of the following groups: thiol (5′-SH), amino (5′-NH2
), phosphate (5′-P), hydroxyl (5′-OH), dimethoxytrityl (5′-DMT) and fluorescein isothiocyanate (5′-FITC). Rup1
are the complementary sequences of up1
, respectively. All the oligonucleotides were desalted and purified by HPLC by the manufacturer (Eurogentec). The purity level of all oligonucleotides was systematically checked by HPLC before use.
Nucleic acid preparation: (ii) 5′-end radiolabelled oligonucleotides
Primers Rup1 and Rup2 were enzymatically phosphorylated with [γ-32P]ATP (Amersham Pharmacia, Buckinghamshire, UK) using the bacteriophage T4 polynucleotide kinase (New England Biolabs, Beverly, MA). Excess [γ-32P]ATP was removed with a chroma-spin column TE-10 (Clontech, Palo Alto, CA).
Nucleic acid preparation: (iii) DNA templates
Fragments of human genomic DNA corresponding to the receptor for advanced glycation end-products (RAGE; GenBank accession no. D28769) were generated by PCR using PCR primers located at 6550–7403 (exon 1–2 template: E1-2) and 8400–9266 (intron 8 template: I8). The sequences of the PCR primers contain the primer sequences of up1 or up2 without the 10 Ts as follows: for E1-2, AGAAGGAGAAGGAAAGGGAAAGGGGCGGCCGCTCGCCTGGTTCTGGAAGACA and CACCAACCCAAACCAACCCAAACCGCGGCCGCTGAGGCCAGTGGAAGTCAGA; and for I8, AGAAGGAGAAGGAAAGGGAAAGGGAGCTGAGGAGGAAGAGAGG and CACCAACCCAAACCAACCCAAACCGAGCTCAAGGCAGGCTAGGAGCTGAG.
Nucleic acid preparation: (iv) labelled nucleic acid probes
RAGE DNA probes were labelled radioactively or with digoxygenin by PCR using PCR primers located at 6550–7091 for probe E1-2 and primers located at position 8400–9266 for the I8 template. Primers for probe E1-2, CCTGTGACAAGACGACTGAA and AGTGAGACGGAGTGTCAGGA; primers for probe I8, AAGGCAGGCTAGGAGCTGAG and AGGGAACTTGACAAGACCGGAG.
Probes were labelled with digoxygenin using a commercial labelling mix (Boehringer-Mannheim, Mannheim, Germany). Probes were radiolabelled by PCR with [α-32P]dCTP (Amersham Pharmacia).
Attachment of oligonucleotides to the glass surface: (i) glass slide pre-treatment
Glass slides were soaked in basic Helmanex solution (HelmanexIIR, 0.25%, 1 N NaOH) for 1 h (HelmanexIIR, Hellma, Müllhein/Baden, Germany), rinsed with water, immersed overnight in 1 N HCl, rinsed again in water and treated for 1 h in sulfuric acid solution (H2SO4/H2O, 1/1, v/v) (H2SO4 95–97%) with a small amount of fresh ammonium persulfate added (BioRad). Acid-treated slides were submitted to extensive washes in water followed by rinsing in ethanol, dried and stored under vacuum for further use.
Attachment of oligonucleotides to the glass surface: (ii) silanisation of glass slide surface
Pre-treated glass slides were immersed into a 5% solution of amino-silane reagents, aminopropyltriethoxysilane (ATS, Aldrich Chemicals, Steinheim, Germany), bis(trimethoxysilylpropyl)amine (BTS, ABCR, Karlsruhe, Germany) or 3-mercaptopropyltrietoxysilane (SHS) in dry acetone. Silanisation was carried out at room temperature for 1 h. After three washes in acetone (5 min/wash), the slides were rinsed once in ethanol, dried and stored under vacuum. We refer to amino-silanised glass slides as ‘derivatised’ glass slides.
Attachment of oligonucleotides to the glass surface: (iii) EDC attachment
Oligonucleotides were prepared as a 1 µM solution in 10 mM 1-methyl-imidazole (pH 7.0) (Sigma Chemicals, Deisenhofen, Germany) containing 40 mM EDC (Pierce, Rockford, IL). Drops of 1–5 µl were spotted on the amino-silanised glass slides. During the 2 h of attachment, the glass slides were kept under a humid atmosphere to avoid evaporation of the drops. The glass slides were washed twice (5 min each) in 5× SSC buffer (0.75 M NaCl, 0.075 M Na citrate pH 7) with 0.1% Tween 20 (Fluka Chemicals) and rinsed in 5× SSC. Slides were finally washed (10 min) in 1.5 M NaCl, 10 mM NaH2
(pH 7) to remove non-covalently attached oligonucleotides (13
), rinsed and stored in 5× SSC at 4°C for further use.
Attachment of oligonucleotides to the glass surface: (iv) cross-linkers chemistry
Functionalisation of silanised glass slides with heterobifunctional cross-linkers. Cross-linker refers to a heterobifunctional cross-linking reagent. Functionalised glass slides refer to a derivatised glass surface on which the cross-linker has been covalently attached by one end. Water soluble cross-linkers were prepared at 20 mM in PBS (0.1 M NaH2PO4, 0.15 M NaCl, pH 7.2). The corresponding non-water-soluble reagents were prepared at 20 mM in DMSO:DMF (9:1 v/v). Derivatised glass slides were covered with the cross-linker solution, protected with a clean micro cover glass (Esco, Erie Scientific, Portsmouth, NH) and reacted for 5 h at 20°C. Glass slides functionalised with water soluble cross-linkers were rinsed in PBS, briefly immersed in water and rinsed in ethanol. Glass slides functionalised with non-water soluble cross-linkers were rinsed in ethanol. Functionalised slides were dried and stored under vacuum for further use. As SIAB and s-SIAB are light sensitive, preparation of SIAB functionalised glass slides, storage and attachment of oligonucleotides were done in the dark.
Oligonucleotide attachment on cross-linker-functionalised glass slides. Oligonucleotides were prepared as 50 µM solutions in saline buffer (NaPi: 0.1 M NaH2PO4 pH 6.5, 0.15 M NaCl) and drops (1–5 µl) were spotted on functionalised glass slides. A pH 6.5 solution was necessary to limit disulfide bond formation between 5′-SH modified oligonucleotides. During the 5 h of attachment at room temperature, the glass slides were kept under a humid atmosphere to avoid evaporation. The glass slides were washed twice (5 min each) on a shaker in NaPi, then a solution of mercaptoethanol (Sigma Chemicals), 10 mM in NaPi was used to cap residual maleimide active moieties (1 h treatment). Glass slides were washed (5 min) in NaPi buffer and submitted to 10 min exposure to a 1.5 M NaCl solution (10 mM NaH2PO4, pH 7) to eliminate non-specific attachment. They were washed in 5× SSC buffer with 0.1% Tween (5 min), rinsed in 5× SSC and stored in 5× SSC at 4°C for further use.
Cover slips were used for quantitative analyses relative to loading density determination (using radioactive hybridisation assay) and attachment was performed on both sides.
Solid phase DNA amplification
Glass slides on which oligonucleotides were covalently bonded were subjected to a 1 h blocking step using 5× SSC buffer containing 0.1% Tween 20, 0.1% BSA (New England Biolabs), washed in 5× SSC buffer and finally washed in water to remove salts. DNA amplification was initiated on glass slides with a PCR mix containing 1× PCR buffer, the four dNTPs at 0.1 mM each, 0.1% BSA, 0.1% Tween supplemented with 1 nM DNA and ampliTaq Gold polymerase (0.05 U/ml, PE Biosystems, Foster City, CA). Cover slip glass slides (4 mm diameter) were immersed into 100 µl of the PCR mix placed into PCR tubes (200 µl, MicroAmpTM, PE Biosystems). For the microscope glass slides, the PCR mix was placed in a frame seal chamber (MJ Research, Waterton, MA). The tubes and the microscope glass slides were put into the 16×16 twin tower block thermocycler (PTC 200, MJ Research). Thermocycling was carried out as follows: 94°C for 9 min and 50 cycles (94°C for 45 s, 65°C for 3 min and 72°C for 4 min). Glass slides were washed in 5× SSC, 0.1% Tween 20 (2 × 10 min) and rinsed and stored in 5× SSC until used.
Radioactively or fluorescently labelled oligonucleotide probes (0.5 µM in 5× SSC, 0.1% Tween 20) were hybridised onto glass slides for 2 h at room temperature. Following hybridisation, the glass slides were rinsed on a shaker in 5× SSC for 5 min and twice in 2× SSC, 0.1% SDS for 5 min at 37°C. Long probes, radioactively-labelled or labelled with digoxygenin, (1 nM in Easyhyb, Boehringer-Mannheim GmbH) were applied to glass slides and hybridised on the MJR PCR machine (95°C for 5 min, 0.1°C/min to 50°C, 50°C for ever). Glass slides were rinsed at 37°C twice in 2× SSC, 0.1% SDS for 15 min, once in 0.2× SSC, 0.1% SDS and twice in 2× SCC. Fluorescent signal of the probe labelled with digoxygenin is obtained by a sandwich assay using an anti-digoxygenin mouse antibody (Boehringer-Mannheim) and a FITC labelled sheep anti mouse (Boehringer-Mannheim).
The amount of hybridisation of the radiolabelled probe was assayed by scintillation counting (Beckman Instruments, Fullerton, CA). The amount of fluorescently labelled probe was detected using the epi-fluorescence microscope setup. To protect the fluorescent labelling dye against photobleaching during irradiation, the glass surface is overlaid with the ProLongTM antifade mix solution (Molecular Probes, Eugene, OR).