Utilizing multiplex fluor LAMPs to illuminate multiple gene expressions in situ
Autoři:
Diona Podushkina aff001; Nick W. West aff001; Edward M. Golenberg aff001
Působiště autorů:
Department of Biological Sciences, Wayne State University, Detroit, Michigan, United States of America
aff001
Vyšlo v časopise:
PLoS ONE 14(10)
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pone.0223333
Souhrn
In situ gene expression detection is the best way to determine temporal and spatial differences in gene expression. However, in situ hybridization procedures are inherently difficult to execute and typically suffer from degradation of sample tissues, limited sensitivity to genes with low expression, high background, and limitation to single gene detections. We propose to utilize an isothermal gene amplification technique, LAMP (Loop-Mediated Isothermal Amplification), to solve these problems in a novel way. LAMP greatly amplifies the signal of expressed genes and can use multiple sets of primers and different fluorescent-labeled probes to produce multiplex gene detection. LAMP is a rapid, isothermal reaction that reduces the handling and degradation of tissue by cutting down on the washing steps required by other methods. Using this technique, we have successfully amplified 3 target genes, have produced positive fluorescent in situ results simultaneously for two genes. We have also demonstrated that LAMP can be used to exploit standard NBT/BCIP (nitro-blue tetrazolium chloride/5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt) detection of single expression. In situ LAMP is a robust and applicable method that can be exploited for detection of gene expression in plant species, as well as in animals and bacteria.
Klíčová slova:
Gene expression – Gene amplification – Probe hybridization – Inflorescences – Antisocial behavior – In situ hybridization – RNA amplification – Spinach
Zdroje
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