Utilizing multiplex fluor LAMPs to illuminate multiple gene expressions in situ

PLoS One. 2019 Oct 4;14(10):e0223333. doi: 10.1371/journal.pone.0223333. eCollection 2019.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA Primers
  • Fluorescent Antibody Technique
  • Fluorescent Dyes
  • Gene Expression Profiling / methods*
  • Gene Expression*
  • Immunohistochemistry
  • In Situ Hybridization / methods*
  • Nucleic Acid Amplification Techniques / methods*
  • Sensitivity and Specificity

Substances

  • DNA Primers
  • Fluorescent Dyes

Grants and funding

This work was supported by a Wayne State University Undergraduate Research Opportunity Award 062018 to D.P. and E.M.G. The work was partially supported by NSF Directorate for Biological Sciences grant 1838291 to E.M.G. (Co-PI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.