SummaryMicroarray 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 mg of total RNA and 1±2 mg of mRNA is required for each hybridisation, which is equivalent to 50±100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of microarrays for transcript pro®ling in speci®c tissues and cell types during plant development. Here we report on a robust and reliable target ampli®cation method that enables transcript pro®ling from sub-mg amounts of plant tissue. Using 0.1 mg of total RNA we show that twofold expression differences are possible to distinguish with 99% con®dence. We also demonstrate the application of this method in an analysis of secondary phloem development in hybrid aspen using de®ned tissue sections, corresponding to 2±4 cell layers with a fresh weight of~0.5 mg.
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. Two types of plasmids were used, one conventional DNA plasmid containing a cytomegalovirus promoter and one suicidal DNA plasmid encoding the Semliki Forest virus (SFV) replicase. RNA, encoding the SFV replicase and the relevant antigen, was delivered either as naked RNA or packaged in SFV suicide particles. In general, the antibody responses induced by the DNA plasmids were low and peaking after three injections, the conventional plasmid giving the highest responses. Also the RNA delivered in SFV particles consistently induced antibody responses, although comparatively low. Analyses of the ratio of immunoglobulin (Ig)G1/IgG2a subclasses in the responses indicated that all plasmids resulted in a bias for a Th2-type of response, while the SFV-particles elicited a Th1 type of response. Importantly, all these immunogens induced an immunological memory, which could be efficiently activated by a booster injection with the corresponding protein, with unchanged patterns of IgG subclasses.
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. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro.
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