Flow-FISH

Flow-FISH (fluorescence in-situ hybridization) is a cytogenetic technique to quantify the copy number of RNA or specific repetitive elements in genomic DNA of whole cell populations via the combination of flow cytometry with cytogenetic fluorescent in situ hybridization staining protocols.[1][2][3]

Flow-FISH is most commonly used to quantify the length of telomeres, which are stretches of repetitious DNA (hexameric TTAGGG repeats) at the distal ends of chromosomes[4] in human white blood cells, and a semi-automated method for doing so was published in Nature Protocols.[1] Telomere length in white blood cells has been a subject of interest because telomere length in these cell types (and also of other somatic tissues) declines gradually over the human lifespan, resulting in cell senescence, apoptosis,[5] or transformation.[6] This decline has been shown to be a surrogate marker for the concomitant decline in the telomere length of the hematopoietic stem cell pool, with the granulocyte lineage giving the best indication, presumably due to the absence of a long lived memory subtype and comparatively rapid turnover of these cells.[7]

Flow-FISH is also suitable for the concomitant detection of RNA and protein.[2] This allows for the identification of cells that not only express a gene, but also translate it into protein. This type of Flow-FISH has been used to study latent infection of viruses such as HIV-1 and EBV,[8][9] but also to track single cell gene expression and translation into protein.[2][10]

  1. ^ a b Baerlocher GM, Vulto I, de Jong G, Lansdorp PM. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc 2006; 1:2365–2376.
  2. ^ a b c Porichis, Filippos; Hart, Meghan G.; Griesbeck, Morgane; Everett, Holly L.; Hassan, Muska; Baxter, Amy E.; Lindqvist, Madelene; Miller, Sara M.; Soghoian, Damien Z.; Kavanagh, Daniel G.; Reynolds, Susan (December 2014). "High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry". Nature Communications. 5 (1): 5641. Bibcode:2014NatCo...5.5641P. doi:10.1038/ncomms6641. ISSN 2041-1723. PMC 4256720. PMID 25472703.
  3. ^ Baxter, Amy E.; Niessl, Julia; Fromentin, Rémi; Richard, Jonathan; Porichis, Filippos; Massanella, Marta; Brassard, Nathalie; Alsahafi, Nirmin; Routy, Jean-Pierre; Finzi, Andrés; Chomont, Nicolas (October 2017). "Multiparametric characterization of rare HIV-infected cells using an RNA-flow FISH technique". Nature Protocols. 12 (10): 2029–2049. doi:10.1038/nprot.2017.079. ISSN 1750-2799. PMC 5697908. PMID 28880280.
  4. ^ Moyzis, R.K. et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc. Natl. Acad. Sci. USA 85, 6622–6626 (1988).
  5. ^ Harley, C.B., Futcher, A.B. & Greider, C.W. Telomeres shorten during ageing of human fibroblasts. Nature 345, 458–460 (1990).
  6. ^ Chang, S., Khoo, C.M., Naylor, M.L., Maser, R.S. & DePinho, R.A. Telomere-based crisis: functional differences between telomerase activation and ALT in tumor progression. Genes Dev. 17, 88–100 (2003).
  7. ^ Rufer N, Brummendorf TH, Kolvraa S, et al. Telomere fluorescence measurements in granulocytes and T lymphocyte subsets point to a high turnover of hematopoietic stem cells and memory T cells in early childhood. J Exp Med 1999; 190:157–167.
  8. ^ Grau-Expósito, Judith; Luque-Ballesteros, Laura; Navarro, Jordi; Curran, Adrian; Burgos, Joaquin; Ribera, Esteban; Torrella, Ariadna; Planas, Bibiana; Badía, Rosa; Martin-Castillo, Mario; Fernández-Sojo, Jesús (2019-08-19). Swanstrom, Ronald (ed.). "Latency reversal agents affect differently the latent reservoir present in distinct CD4+ T subpopulations". PLOS Pathogens. 15 (8): e1007991. doi:10.1371/journal.ppat.1007991. ISSN 1553-7374. PMC 6715238. PMID 31425551.
  9. ^ Fournier, Benjamin; Boutboul, David; Bruneau, Julie; Miot, Charline; Boulanger, Cécile; Malphettes, Marion; Pellier, Isabelle; Dunogué, Bertrand; Terrier, Benjamin; Suarez, Felipe; Blanche, Stéphane (2020-11-02). "Rapid identification and characterization of infected cells in blood during chronic active Epstein-Barr virus infection". Journal of Experimental Medicine. 217 (11): e20192262. doi:10.1084/jem.20192262. ISSN 0022-1007. PMC 7596820. PMID 32812031.
  10. ^ Nicolet, Benoit P.; Guislain, Aurelie; Wolkers, Monika C. (2017-01-15). "Combined Single-Cell Measurement of Cytokine mRNA and Protein Identifies T Cells with Persistent Effector Function". The Journal of Immunology. 198 (2): 962–970. doi:10.4049/jimmunol.1601531. ISSN 0022-1767. PMID 27927969.