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Flow-FISH(fluorescence in-situ hybridization) is acytogenetictechnique to quantify the copy number ofRNAor specific repetitive elements in genomicDNAof whole cell populations via the combination offlow cytometrywith cytogeneticfluorescent in situ hybridizationstaining protocols.[1][2][3]

Flow-FISH is most commonly used to quantify the length oftelomeres,which are stretches of repetitiousDNA(hexameric TTAGGG repeats) at the distal ends ofchromosomes[4]in humanwhite blood cells,and a semi-automated method for doing so was published inNature Protocols.[1]Telomerelength inwhite blood cellshas been a subject of interest because telomere length in these cell types (and also of othersomatictissues) declines gradually over the human lifespan, resulting in cellsenescence,apoptosis,[5]ortransformation.[6]This decline has been shown to be a surrogate marker for the concomitant decline in the telomere length of thehematopoietic stem cellpool, with thegranulocytelineage 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 ofRNAandprotein.[2]This allows for the identification of cells that not onlyexpress a gene,but alsotranslateit intoprotein.This type of Flow-FISH has been used to studylatent infectionofvirusessuch asHIV-1andEBV,[8][9]but also to track single cellgene expressionandtranslationinto protein.[2][10]

Q-FISH to flow-FISH

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Flow-FISH was first published in 1998 by Rufer et al.[11]as a modification of another technique for analyzing telomere length,Q-FISH,that employspeptide nucleic acidprobes[12]of a 3'-CCCTAACCCTAACCCTAA-5' sequence labeled with afluorescinfluorophoreto stain telomeric repeats on preparedmetaphasespreads of cells that have been treated withcolcemid,hypotonic shock, andfixationto slides viamethanol/acetic acidtreatment[13]Images of the resultant fluorescent spots could then be analyzed via a specialized computer program to yield quantitative fluorescence values that can then be used to estimate actual telomere length. The fluorescence yielded by probe staining is considered to be quantitative becausePNAbinds preferentially to DNA at low ionic salt concentrations and in the presence offormamide,thus the DNA duplex may not reform once it has been melted and annealed toPNAprobe, allowing the probe to saturate its target repeat sequence (as it is not displaced from the target DNA by competing anti sense DNA on the complementary strand), thus yielding a reliable and quantifiable readout of the frequency ofPNAprobe target at a given chromosomal site after washing away of unbound probe.[13]

DNA is denatured via heat and formamide treatment, PNA probe is allowed to hybridize, excess probe is washed away, reading is taken in flow cytometer

Innovation

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Unlike Q-FISH, Flow-FISH utilizes the quantitative properties of telomere specificPNAprobe retention to quantify median fluorescence in a population of cells, via the use of aflow cytometer,instead of a fluorescence microscope.[14]The primary advantage of this technique is that it eliminates the time required in Q-FISH to prepare metaphase spreads of cells of interest, and that flow cytometric analysis is also considerably faster than the methods required to acquire and analyze Q-FISH prepared slides. Flow-FISH thus allows for a higher throughput analysis of telomere length in blood leukocytes, which are a readily available form of human tissue sample. The most recent versions of the flow-FISH technique include an internal control population of cow thymocytes with a known telomere length detected by TRF or telomere restriction fragment analysis to which the fluorescence of a given unknown sample may be compared. Because cow thymocytes take up LDS751 dye to a lesser extent than their human counterparts, they may be reliably differentiated via plotting and gating the desired populations. Other cell types that have not in the past proven to be good candidates for flow-FISH can be analyzed via extraction of nuclei and performance of the technique on them directly.[15]

Multiparametric analysis allows for differentiation of cell types within one sample, allowing for internal control, and analysis of leukocyte subtypes.

References

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  1. ^abBaerlocher 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. ^abcPorichis, 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.ISSN2041-1723.PMC4256720.PMID25472703.
  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.ISSN1750-2799.PMC5697908.PMID28880280.
  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.ISSN1553-7374.PMC6715238.PMID31425551.
  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.ISSN0022-1007.PMC7596820.PMID32812031.
  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.ISSN0022-1767.PMID27927969.
  11. ^Rufer, N., Dragowska, W., Thornbury, G., Roosnek, E. & Lansdorp, P.M. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nature Biotechnol. 16, 743–747 (1998).
  12. ^Egholm, M. et al. PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules. Nature 365, 566–568 (1993).
  13. ^abLansdorp, P.M. et al. Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet. 5, 685–691 (1996).
  14. ^Baerlocher, G.M. & Lansdorp, P.M. Telomere length measurements in leukocyte subsets by automated multicolor flow FISH. Cytometry A 55, 1–6 (2003).
  15. ^Wieser, M. et al. Nuclear flow FISH: isolation of cell nuclei improves the determination of telomere lengths. Exp. Gerontol. 41, 230–235 (2006).
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