Assessment of 35mer amino-modified oligonucleotide based microarray with bacterial samples

• Published in: Journal of microbiological methods 2004-05, Vol.57 (2), p.207-218
• Main Authors: Calevro, Federica, Charles, Hubert, Reymond, Nancie, Dugas, Vincent, Cloarec, Jean-Pierre, Bernillon, Jacques, Rahbé, Yvan, Febvay, Gérard, Fayard, Jean-Michel
• Format: Article
• Language: English
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Base Pair Mismatch; Biological and medical sciences; Buchnera; Buchnera - genetics; Buchnera aphidicola; DNA, Complementary; DNA, Complementary - chemistry; DNA, Complementary - genetics; Ecology, environment; Fluorescence; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Profiling; Gene Expression Profiling - methods; Intracellular bacteria; Life Sciences; Microbiology; Nucleic Acid Hybridization; Nucleic Acid Hybridization - methods; Oligonucleotide Array Sequence Analysis; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotide microarrays; Oligonucleotide Probes; RNA, Bacterial; RNA, Bacterial - genetics; RNA, Bacterial - isolation & purification; Sensitivity and Specificity; Symbiosis
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2004.01.009

Parallel quantification of a large number of messenger RNA transcripts, using microarray technology, promises to provide unsuspected information about many cellular processes. Although experimental protocols on microarray applications are available, only limited methodological information on glass-slide manufacturing and signal interpretation has been published. The aim of this paper is to provide new insights into the practical aspects of the construction and hybridization of oligonucleotide-based microarrays. The intracellular symbiotic bacterium of aphids, Buchnera aphidicola, is used here as a model organism. The first part of the work is devoted to the optimization of procedures for printing slides, labeling of cDNA targets and hybridization. In the second part, based on a statistical analysis of the results, we discuss the influence of the probe attachment chemistry, of the labeling method, of the oligonucleotide position and of the concentration of a spotted oligonucleotide on signal intensity. The problem of signal specificity is also addressed, based on the calculation of the fluorescent ratio for each probe to its corresponding mismatch control probe. Lastly, the selection of internal spiked RNAs appropriate to our bacterial samples and useful for the data normalization step is presented.