Abstract
BackgroundAlzheimer's disease (AD) is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels. Complementary to this approach, we described here a novel technology designed specifically to isolate rare and novel genes previously undetectable by other methods. We have used this method to identify differentially expressed genes in brains affected by AD. Our method, termed Subtractive Transcription-based Amplification of mRNA (STAR), is a combination of subtractive RNA/DNA hybridization and RNA amplification, which allows the removal of non-differentially expressed transcripts and the linear amplification of the differentially expressed genes.ResultsUsing the STAR technology we have identified over 800 differentially expressed sequences in AD brains, both up- and down- regulated, compared to age-matched controls. Over 55% of the sequences represent genes of unknown function and roughly half of them were novel and rare discoveries in the human brain. The expression changes of nearly 80 unique genes were further confirmed by qRT-PCR and the association of additional genes with AD and/or neurodegeneration was established using an in-house literature mining tool (LitMiner).ConclusionThe STAR process significantly amplifies unique and rare sequences relative to abundant housekeeping genes and, as a consequence, identifies genes not previously linked to AD. This method also offers new opportunities to study the subtle changes in gene expression that potentially contribute to the development and/or progression of AD.
Highlights
Alzheimer's disease (AD) is a complex disorder that involves multiple biological processes
The major disadvantages of this method include its lack of sensitivity for the detection of rare RNA species, the high number of false positives generated during PCR and cloning of the differentially expressed products from low resolution polyacrylamide gels, where an apparent single band may contain multiple cDNA species
The Subtractive Transcription-based Amplification of mRNA (STAR) process significantly increases the levels of unique and rare sequences relative to abundant housekeeping genes and identified changes in the expression of genes not previously linked to the AD
Summary
Alzheimer's disease (AD) is a complex disorder that involves multiple biological processes. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels Complementary to this approach, we described here a novel technology designed to isolate rare and novel genes previously undetectable by other methods. The most commonly used technology for the assessment of gene expression changes in postmortem brain is the DNA microarray [1,2,3,4,5] This approach has confirmed the involvement of genes implicated in AD by conventional methods, and revealed changes in additional genes, not previously associated with AD [6,7] As this method requires a priori knowledge of gene sequences, it cannot be applied as a discovery tool for novel transcripts. Differential display is labor intensive and unreliable for this application
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