Microarrays--the challenge of preparing brain tissue samples.
ABSTRACT Microarray experiments allow researchers to collect an amazing amount of gene expression data that have the potential to provide unique information to help interpretation of the biological functions of the central nervous system. These experiments are, however, technically demanding and present unique difficulties when used in the context of neuroscience research, in particular. Success or failure of microarray experiments are highly dependent on reproducible target preparations. This involves a relatively long chain of preparation steps, such as removal of tissue from experimental animals or from post-mortem human brains, storage, selection, and excision of brain regions. This is followed by RNA extraction, reverse transcription, and labeling of target cDNAs or cRNAs. Additionally, it is emphasized that the quality of microarray data largely relies on the proper handling of animals throughout experiments and the time of the day when experiments are stopped. This article tries to provide hints for some basic rules to be observed in preparation of samples for expression profiling studies.
- SourceAvailable from: ncbi.nlm.nih.gov[show abstract] [hide abstract]
ABSTRACT: Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.Journal of Molecular Diagnostics 06/2000; 2(2):84-91. · 3.95 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A cell's structural and functional characteristics are dependent on the specific complement of genes it expresses. The ability to study and compare gene usage at the cellular level will therefore provide valuable insights into cell physiology. Such analyses are complicated by problems associated with sample collection, sample size and the limited sensitivity of expression assays. Advances have been made in approaches to the collection of cellular material and the performance of single-cell gene expression analysis. Recent development in global amplification of mRNA may soon permit expression analyses of single cells to be performed on DNA microarrays.Current Opinion in Biotechnology 01/2000; 10(6):579-82. · 7.86 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Rapid degradation of c-fos proto-oncogene mRNA is crucial for transient c-fos gene expression. Experiments were performed to investigate the cellular mechanisms responsible for the extremely short half-life of human c-fos mRNA in growth-factor-stimulated fibroblasts. These experiments demonstrate the existence of two distinct cellular pathways for rapid c-fos mRNA degradation. Each of these pathways recognizes a different, functionally independent instability determinant within the c-fos transcript. One instability determinant, which is located within the c-fos 3'-untranslated region, is a 75-nucleotide AU-rich segment. Insertion of this element into beta-globin mRNA markedly reduces the half-life of that normally long-lived message. Nevertheless, specific deletion of the AU-rich element from c-fos mRNA has little effect on the transcript's cytoplasmic half-life due to the presence of the other c-fos instability determinant, which is located in the protein-coding segment of the c-fos message. Examination of mRNA decay in cells treated with transcription inhibitors indicates that one c-fos mRNA degradation pathway is dependent on RNA synthesis, whereas the other is not.Genes & Development 02/1989; 3(1):60-72. · 12.44 Impact Factor