Assessment of 35mer amino-modified oligonucleotide based microarray with bacterial samples.
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.
Calevro
Federica
F
UMR INRA/INSA 203, Laboratoire de Biologie Fonctionnelle, Insectes et Interactions, INSA-Lyon, 20 Avenue Albert Einstein, 69621 Villeurbanne Cedex, France. Federica.Calevro@insa-lyon.fr
Charles
Hubert
H
Reymond
Nancie
N
Dugas
Vincent
V
Cloarec
Jean-Pierre
JP
Bernillon
Jacques
J
Rahbé
Yvan
Y
Febvay
Gérard
G
Fayard
Jean-Michel
JM
D020137:Base Pair Mismatch; D020635:Buchnera / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005453:Fluorescence; D020869:Gene Expression Profiling / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial