title: 16S and 23S rRNA Gel Pad Microarray
status: Public on Oct 22 2005
contact.city: Louvain-la-Neuve
contact.first: Laurent
contact.last: Eyers
contact.email: eyers@gebi.ucl.ac.be
contact.address: Place Croix du Sud 2/19
contact.department: Unit of bioengineering
contact.country: Belgium
contact.institute: Catholic University of Louvain
organism: unidentified
description: Probes targeting the 16S and 23S rRNA of various organisms (mainly Bacteria)
manufacture_protocol: Gel-Pad microarrays consisted of 100 x 100 x 20 µm polyacrylamide gel pads photopolymerised to a glass slide with a center-to-center distance of 300 µm (Guschin et al. 1997, Anal. Biochem. 250:203-211). After activation of gel pads (Proudnikov et al. 1998, Anal. Biochem. 259:34-41), oligonucleotide probes were spotted (2 pmol deposited) on each gel pad by Schiff base coupling to aldehyde groups within the gel pads (Bavykin et al. 2001, Appl. Environ. Microbiol. 67:922-928). The coupling was stabilised by reduction with NaCNBH3 (Proudnikov et al. 1998, Anal. Biochem. 259:34-41). The design of oligonucleotide probes targeting rRNA molecules was done as described previously (El Fantroussi et al. 2003, Appl. Environ. Microbiol. 69:2377-2382). Gel-Pad microarrays contained 29 different perfect match oligonucleotide probes (13-25 nucleotides in length) that were complementary to universal, domain, species, and phylogenetic group specific rRNA sequences. In addition to perfect match probes, single mismatch probes were printed on the Gel-Pad microarray. Design of single mismatch probes was based on several criteria: i) GC content was not changed (e.g., substitution of G to C (c-MM probes) or C to G (g-MM probes)), ii) mismatches at terminal positions were avoided (Urakawa et al. 2002, Appl. Environ. Microbiol. 68:235-244), iii) non-complementarity of MM probes to any microorganism was checked by comparing MM probe sequences to the 1999 alignment of 16S rRNA sequences with ARB software (http://www.mikro.biologie.tu-muenchen.de) and the GenBank database (http://www.ncbi.nih.gov/Genbank), and iv) approximately 4°C differences in theoretical Tds between PM and c-MM and about 6°C differences between PM and g-MM were chosen.