Flow-FISH (fluorescent in-situ hybridization) is a

cytogenetic Cytogenetics is essentially a branch of genetics, but is also a part of cell biology/cytology (a subdivision of human anatomy), that is concerned with how the chromosomes relate to cell behaviour, particularly to their behaviour during mitosis an ...

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.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. Flow-FISH is most commonly used to quantify the length of

telomeres A telomere (; ) is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Although there are different architectures, telomeres, in a broad sense, are a widespread genetic feature mos ...

, which are stretches of repetitious DNA (hexameric TTAGGG repeats) at the distal ends of

chromosomes A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...

in human

white blood cells White blood cells, also called leukocytes or leucocytes, are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. All white blood cells are produced and derived from mult ...

, and a semi-automated method for doing so was published in

Nature Protocols ''Nature Protocols'', published by the Nature Publishing Group, is an on-line scientific journal publishing methods in a recipe-style format. The journal was launched in June 2006 and the content includes both classical methods and cutting-edge te ...

Telomere A telomere (; ) is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Although there are different architectures, telomeres, in a broad sense, are a widespread genetic feature mos ...

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, or

transformation Transformation may refer to: Science and mathematics In biology and medicine * Metamorphosis, the biological process of changing physical form after birth or hatching * Malignant transformation, the process of cells becoming cancerous * Tran ...

. 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. Flow-FISH is also suitable for the concomitant detection of RNA and

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...

. This allows for the identification of cells that not only express a gene, but also

translate Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transl ...

it into

. This type of Flow-FISH has been used to study latent infection of

virus A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea. Since Dmitri Ivanovsk ...

es such as

HIV-1 The subtypes of HIV include two major types, HIV type 1 (HIV-1) and HIV type 2 (HIV-2). HIV-1 is related to viruses found in chimpanzees and gorillas living in western Africa, while HIV-2 viruses are related to viruses found in the sooty mangabey ...

and EBV, but also to track single cell gene expression and

translation Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text. The English language draws a terminological distinction (which does not exist in every language) between ''transla ...

into protein.

Q-FISH to flow-FISH

Flow-FISH was first published in 1998 by Rufer et al. as a modification of another technique for analyzing telomere length, Q-FISH, that employs

peptide nucleic acid Peptide nucleic acid (PNA) is an artificially synthesized polymer similar to DNA or RNA. Synthetic peptide nucleic acid oligomers have been used in recent years in molecular biology procedures, diagnostic assays, and antisense therapies. Due ...

probes of a 3'-CCCTAACCCTAACCCTAA-5' sequence labeled with a fluorescin

fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...

to stain telomeric repeats on prepared

metaphase Metaphase ( and ) is a stage of mitosis in the eukaryotic cell cycle in which chromosomes are at their second-most condensed and coiled stage (they are at their most condensed in anaphase). These chromosomes, carrying genetic information, alig ...

spreads of cells that have been treated with colcemid, hypotonic shock, and fixation to slides via methanol/ acetic acid treatmentLansdorp, P.M. et al. Heterogeneity in telomere length of human chromosomes. Hum. Mol. Genet. 5, 685–691 (1996). 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 because PNA binds preferentially to DNA at low ionic salt concentrations and in the presence of

formamide Formamide is an amide derived from formic acid. It is a colorless liquid which is miscible with water and has an ammonia-like odor. It is chemical feedstock for the manufacture of sulfa drugs and other pharmaceuticals, herbicides and pesticides, a ...

, thus the DNA duplex may not reform once it has been melted and annealed to PNA probe, 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 of PNA probe target at a given chromosomal site after washing away of unbound probe.

Innovation

Unlike Q-FISH, Flow-FISH utilizes the quantitative properties of telomere specific PNA probe retention to quantify median fluorescence in a population of cells, via the use of a

flow cytometer Flow cytometry (FC) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the fl ...

, instead of a fluorescence microscope. 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.Wieser, M. et al. Nuclear flow FISH: isolation of cell nuclei improves the determination of telomere lengths. Exp. Gerontol. 41, 230–235 (2006).

References

Biochemistry methods Gene expression Flow cytometry Lymphocytes Telomeres