Karl H. Hasenstein, Min Chen
Univ. Louisiana Lafayette, United States of America
Understanding gene expression and regulation especially on a cellular level requires rapid, localized and ideally repeated sampling of mRNA. Current technologies inevitable rely on the extraction of tissue, cytoplasm aspiration, or cell lysates, all of which pose problems because of unspecific sampling, contamination, and lack of repeatability. These methods are destructive and do not distinguish between genomic DNA and RNA. Moreover, extracted mRNA is typically contaminated by extracted cytoplasm, nuclear DNA, or other compounds, and the required purification leads to loss of especially low-abundant mRNA. Biological systems, where all regulatory and response steps take place in a single cell are thus not accessible to localized gene expression studies. This predicament lead to the development of a needle-based mRNA extraction system that provides high temporal and spatial resolution that allows for repeated removal of mRNA from living material. This development (dubbed Solid Phase Gene Extraction, SPGE) can be designed to hybridize with gene-specific or generic (oligo-dT) sequences. It is not species-specific and can be employed to access tissue or single cells. We demonstrate the versatility and validity of this novel RNA extraction technique by simultaneously profiling nanos and bicoid mRNA in individual Drosophila eggs. The distribution of these genes resulted in the same longitudinal distribution as previously described distribution profiles. The low impact of SPGE sampling was underscored by the normal development of repeatedly sampled eggs. Actin isoforms in germinating seedlings varied in the apical tissue of roots as a function of imbibition and permits discrimination between newly synthesized and maternal genes. Gene expression in human tumor tissue suggests that SPGE has the potential to supplement or eliminate biopsies. Examination of several genes that are typically used as references in gene expression studies show that SPGE is more sensitive than tissue-based extraction and has the potential to function as a self-referencing system. These data demonstrate the universality of SPGE as a simple, generic, analytical, and diagnostic procedure.
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