Use of DNA microarrays to study global patterns of gene expression.
DNA microarray technology represents an extremely powerful tool to understand the biology of Myobacterium tuberculosis and its interaction with the host. It opens up the possibility of monitoring the expression level of thousands of genes in parallel, thus the ability to test the effect on global transcription of different experimental conditions. Whole genome microarrays consist either of PCR amplicons or oligonucleotides representing every open reading frame in a genome printed on a slide in a high-density matrix. The gene identity and position of each spot is known and can be tracked.Transcription profiling experiments are designed to compare gene expression in bacteria exposed to two different conditions. The RNA from the two different cultures is extracted and reverse transcribed to obtain differentially labeled cDNA by incorporating dUTP or dCTP conjugated with either Cy5 or Cy3, two fluorophores able to emit fluorescence of two different wavelengths. Equal amounts of the two differentially labeled cDNA are mixed, applied to the array surface, and allowed to hybridize to the corresponding gene-specific target. The microarray is finally scanned to obtain two overlapping images each relative to the fluorescence emitted from each label. The images obtained are then analyzed by several software packages to identify and quantify the spots corresponding with the gene-specific probes. After image processing, the data are normalized and then analyzed to determine those genes whose differential expression between the two samples is statistically significant. However, the statistical analysis of microarray data alone is not usually considered enough to confirm differential expression of a gene, and validation with an independent technique, such as quantitative RT-PCR, is required.
Provvedi
Roberta
R
Department of Biology, University of Padova, Padova, Italy.
Palù
Giorgio
G
Manganelli
Riccardo
R
D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*