Identification and quantitation of gene expression
Northern blot hybridization and reverse-transcription polymerase chain reaction (RT-PCR) are commonly used methodologies for detection and quantitation of gene expression.
Northern blot: standard method for determining transcript size and for comparison of messenger abundance between samples. For Northern blot analysis, RNA is extracted from tumour lysates and size-separated by gel electrophoresis. After transfer to filters, specific RNA species are detected by probe hybridization.
RT-PCR: RNA is first converted into cDNA and specific genes analysed using gene-specific primers in PCR reactions. RT-PCR is extremely sensitive but generally lacks the quantitation of Northern analysis. A complementary approach is RNA in situ hybridization, identifying cellular distribution of RNA sequences within a tumour section, allowing study of tumour heterogeneity and subclone development.
Limitations of Northern blot and RT-PCR: analysis is restricted to only a few genes at a time, and sequence must be known in order to generate probes. Each cell may express 10 000-50 000 genes, so gene expression patterns may be tumour-specific. It has been difficult to monitor expression patterns of thousands of genes simultaneously until recently.
SAGE: Serial Analysis of Gene Expression (SAGE) can be applied in laboratories with PCR and sequencing capabilities. SAGE is based on generations of clones of short sequence tags derived from mRNA extracted from target tissue. Each individual tag represents a single mRNA, so sequencing of concatenates describes the pattern and abundance of mRNAs in target tissue. It is thus possible to screen several thousand tags in a few weeks. Sequences are screened against existing databases for known genes, and previously uncharacterized sequences are analysed in more detail. With SAGE, data are digitized and reusable, so once sequence information is obtained it can be reused and rescreened against any existing or developing genome database.
Bioinformatics: Bioinformatics is the use of computers to analyse biological information; a rapidly developing area of cancer research, given the vast amounts of sequence data produced by molecular biology projects. Nucleotide sequences can be used to interrogate public sequence databases for homology and identification over the internet. They can also be used to identify putative gene regulatory elements and gene features. The establishment of the Cancer Genome Anatomy Project (CGAP) means tumour-specific patterns of gene expression are now being held in databases.
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