Improving reliability and performance of DNA microarrays.

A great many platforms and versions of the microarray technology, with different characteristics and applications, have been developed. This review will describe some key issues in reliability and performance with the two most commonly used platforms for gene expression analysis, in situ-synthesized oligonucleotide microarrays or GeneChips and spotted microarrays. Some recent advances and new applications within the field will be mentioned briefly.

Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation.

Background: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed.

Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method.

Background: Neural stem cells (NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system.

Amplification of mRNA populations by a cDNA tag strategy.

Here we describe an amplification method for global transcript analysis. The strategy relies on amplification of cDNA tags (signature tags) achieved by random fragmentation of the cDNAs to short tags of similar length, isolation of the 3' ends and then PCR amplification of the 3'-end signature tag population. This method minimizes biased amplification that may occur during parallel amplification of long and short templates.

Gene expression analysis by signature pyrosequencing.

We describe a novel method for transcript profiling based on high-throughput parallel sequencing of signature tags using a non-gel-based microtiter plate format. The method relies on the identification of cDNA clones by pyrosequencing of the region corresponding to the 3'-end of the mRNA preceding the poly(A) tail. Simultaneously, the method can be used for gene discovery, since tags corresponding to unknown genes can be further characterized by extended sequencing.

Comparative immunization study using RNA and DNA constructs encoding a part of the Plasmodium falciparum antigen Pf332.

Development of nucleic acid-based vaccines against parasitic diseases shows great promise, although certain concerns about safety aspects of conventional DNA vaccines have been raised. This study presents a comparison of antibody responses induced in mice by DNA and RNA-based immunization with vectors encoding a part of the P. falciparum antigen Pf332.

cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol.

Microarray technology is becoming an important comprehensive tool to study gene expression in plants. However, the use of this technology is limited by the large amount of sample tissue needed for microarray analysis. Generally, 50-200 microg of total RNA and 1-2 microg of mRNA is required for each hybridisation, which is equivalent to 50-100 mg of plant tissue.

Synthesis of fluorescent derivatives of 7-methylguanine through reaction with 2-aryl-substituted malondialdehydes: analysis by HPLC with fluorescence detection.

Fluorescent derivatives of 7-methylguanine were prepared through reaction with 2-aryl-substituted-malondialdehydes and analysed by reversed-phase HPLC with fluorescence detection. Reaction of carbons 1 and 3 of the malondialdehyde molecule at the N1 and N2 positions of 7-methylguanine yielded fluorescent tricyclic structures. Two novel fluorescent derivatives of 7-MeG were obtained, namely, 7-(3,4-dimethoxyphenyl)-10-oxo-1-methyl-9,10-dihydropyrimido[1,2- alpha]purine (yield 15-34%) and 7-(1-naphthyl)-10-oxo-1-methyl-9,10- dihydropyrimido[1,2-alpha]purine (yield 56-70%) after reaction with 3,4-dimethoxyphenylmalondialdehyde and 1-naphthylmalondialdehyde, respectively which were characterized by IR, NMR, MS and UV and fluorescence spectroscopy.