Flow-FISH

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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]

Contents

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]

Q-FISH to flow-FISH

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 employs peptide nucleic acid probes [12] of a 3'-CCCTAACCCTAACCCTAA-5' sequence labeled with a fluorescin fluorophore to stain telomeric repeats on prepared metaphase spreads of cells that have been treated with colcemid, hypotonic shock, and fixation to slides via methanol/acetic acid treatment [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 because PNA binds preferentially to DNA at low ionic salt concentrations and in the presence of formamide, 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. [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 Flow-FISH 1.JPG
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

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, 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. Flow-FISH 2.JPG
Multiparametric analysis allows for differentiation of cell types within one sample, allowing for internal control, and analysis of leukocyte subtypes.

Related Research Articles

<span class="mw-page-title-main">Telomere</span> Region of repetitive nucleotide sequences on chromosomes

A telomere is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Telomeres are a widespread genetic feature most commonly found in eukaryotes. In most, if not all species possessing them, they protect the terminal regions of chromosomal DNA from progressive degradation and ensure the integrity of linear chromosomes by preventing DNA repair systems from mistaking the very ends of the DNA strand for a double-strand break.

<span class="mw-page-title-main">Fluorescent tag</span>

In molecular biology and biotechnology, a fluorescent tag, also known as a fluorescent label or fluorescent probe, is a molecule that is attached chemically to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Generally, fluorescent tagging, or labeling, uses a reactive derivative of a fluorescent molecule known as a fluorophore. The fluorophore selectively binds to a specific region or functional group on the target molecule and can be attached chemically or biologically. Various labeling techniques such as enzymatic labeling, protein labeling, and genetic labeling are widely utilized. Ethidium bromide, fluorescein and green fluorescent protein are common tags. The most commonly labelled molecules are antibodies, proteins, amino acids and peptides which are then used as specific probes for detection of a particular target.

<span class="mw-page-title-main">Telomerase</span> Telomere-restoring protein active in the most rapidly dividing cells

Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres. A telomere is a region of repetitive sequences at each end of the chromosomes of most eukaryotes. Telomeres protect the end of the chromosome from DNA damage or from fusion with neighbouring chromosomes. The fruit fly Drosophila melanogaster lacks telomerase, but instead uses retrotransposons to maintain telomeres.

<span class="mw-page-title-main">Cytogenetics</span> Branch of genetics

Cytogenetics is essentially a branch of genetics, but is also a part of cell biology/cytology, that is concerned with how the chromosomes relate to cell behaviour, particularly to their behaviour during mitosis and meiosis. Techniques used include karyotyping, analysis of G-banded chromosomes, other cytogenetic banding techniques, as well as molecular cytogenetics such as fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH).

Comparative genomic hybridization (CGH) is a molecular cytogenetic method for analysing copy number variations (CNVs) relative to ploidy level in the DNA of a test sample compared to a reference sample, without the need for culturing cells. The aim of this technique is to quickly and efficiently compare two genomic DNA samples arising from two sources, which are most often closely related, because it is suspected that they contain differences in terms of either gains or losses of either whole chromosomes or subchromosomal regions. This technique was originally developed for the evaluation of the differences between the chromosomal complements of solid tumor and normal tissue, and has an improved resolution of 5–10 megabases compared to the more traditional cytogenetic analysis techniques of giemsa banding and fluorescence in situ hybridization (FISH) which are limited by the resolution of the microscope utilized.

<span class="mw-page-title-main">Hoechst stain</span> Fluorescent dye used to stain DNA

Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These bis-benzimides were originally developed by Hoechst AG, which numbered all their compounds so that the dye Hoechst 33342 is the 33,342nd compound made by the company. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation–emission spectra.

Fluorescence <i>in situ</i> hybridization Genetic testing technique

Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescent probes that bind to only particular parts of a nucleic acid sequence with a high degree of sequence complementarity. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific DNA sequences on chromosomes. Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes. FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification. FISH can also be used to detect and localize specific RNA targets in cells, circulating tumor cells, and tissue samples. In this context, it can help define the spatial-temporal patterns of gene expression within cells and tissues.

<i>In situ</i> hybridization Laboratory technique to localize nucleic acids

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acid strand to localize a specific DNA or RNA sequence in a portion or section of tissue or if the tissue is small enough, in the entire tissue, in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.

<span class="mw-page-title-main">Dyskeratosis congenita</span> Medical condition

Dyskeratosis congenita (DKC), also known as Zinsser-Engman-Cole syndrome, is a rare progressive congenital disorder with a highly variable phenotype. The entity was classically defined by the triad of abnormal skin pigmentation, nail dystrophy, and leukoplakia of the oral mucosa, and myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), but these components do not always occur. DKC is characterized by short telomeres. The disease initially can affect the skin, but a major consequence is progressive bone marrow failure which occurs in over 80%, causing early mortality.

<span class="mw-page-title-main">Sugar phosphates</span>

Sugar phosphates are often used in biological systems to store or transfer energy. They also form the backbone for DNA and RNA. Sugar phosphate backbone geometry is altered in the vicinity of the modified nucleotides.

<span class="mw-page-title-main">Molecular cytogenetics</span>

Molecular cytogenetics combines two disciplines, molecular biology and cytogenetics, and involves the analysis of chromosome structure to help distinguish normal and cancer-causing cells. Human cytogenetics began in 1956 when it was discovered that normal human cells contain 46 chromosomes. However, the first microscopic observations of chromosomes were reported by Arnold, Flemming, and Hansemann in the late 1800s. Their work was ignored for decades until the actual chromosome number in humans was discovered as 46. In 1879, Arnold examined sarcoma and carcinoma cells having very large nuclei. Today, the study of molecular cytogenetics can be useful in diagnosing and treating various malignancies such as hematological malignancies, brain tumors, and other precursors of cancer. The field is overall focused on studying the evolution of chromosomes, more specifically the number, structure, function, and origin of chromosome abnormalities. It includes a series of techniques referred to as fluorescence in situ hybridization, or FISH, in which DNA probes are labeled with different colored fluorescent tags to visualize one or more specific regions of the genome. Introduced in the 1980s, FISH uses probes with complementary base sequences to locate the presence or absence of the specific DNA regions. FISH can either be performed as a direct approach to metaphase chromosomes or interphase nuclei. Alternatively, an indirect approach can be taken in which the entire genome can be assessed for copy number changes using virtual karyotyping. Virtual karyotypes are generated from arrays made of thousands to millions of probes, and computational tools are used to recreate the genome in silico.

<span class="mw-page-title-main">Telomerase reverse transcriptase</span> Catalytic subunit of the enzyme telomerase

Telomerase reverse transcriptase is a catalytic subunit of the enzyme telomerase, which, together with the telomerase RNA component (TERC), comprises the most important unit of the telomerase complex.

<span class="mw-page-title-main">Telomeric repeat-binding factor 2</span> Protein

Telomeric repeat-binding factor 2 is a protein that is present at telomeres throughout the cell cycle. It is also known as TERF2, TRF2, and TRBF2, and is encoded in humans by the TERF2 gene. It is a component of the shelterin nucleoprotein complex and a second negative regulator of telomere length, playing a key role in the protective activity of telomeres. It was first reported in 1997 in the lab of Titia de Lange, where a DNA sequence similar, but not identical, to TERF1 was discovered, with respect to the Myb-domain. De Lange isolated the new Myb-containing protein sequence and called it TERF2.

<span class="mw-page-title-main">Telomerase RNA component</span> NcRNA found in eukaryotes

Telomerase RNA component, also known as TR, TER or TERC, is an ncRNA found in eukaryotes that is a component of telomerase, the enzyme used to extend telomeres. TERC serves as a template for telomere replication by telomerase. Telomerase RNAs differ greatly in sequence and structure between vertebrates, ciliates and yeasts, but they share a 5' pseudoknot structure close to the template sequence. The vertebrate telomerase RNAs have a 3' H/ACA snoRNA-like domain.

A Riboprobe, abbreviation of RNA probe, is a segment of labelled RNA that can be used to detect a target mRNA or DNA during in situ hybridization. RNA probes can be produced by in vitro transcription of cloned DNA inserted in a suitable plasmid downstream of a viral promoter. Some bacterial viruses code for their own RNA polymerases, which are highly specific for the viral promoters. Using these enzymes, labeled NTPs, and inserts inserted in both forward and reverse orientations, both sense and antisense riboprobes can be generated from a cloned gene.

Chromogenic in situ hybridization (CISH) is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization. It was developed around the year 2000 as an alternative to fluorescence in situ hybridization (FISH) for detection of HER-2/neu oncogene amplification. CISH is similar to FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA. However, CISH is much more practical in diagnostic laboratories because it uses bright-field microscopes rather than the more expensive and complicated fluorescence microscopes used in FISH.

Quantitative Fluorescent in situ hybridization (Q-FISH) is a cytogenetic technique based on the traditional FISH methodology. In Q-FISH, the technique uses labelled synthetic DNA mimics called peptide nucleic acid (PNA) oligonucleotides to quantify target sequences in chromosomal DNA using fluorescent microscopy and analysis software. Q-FISH is most commonly used to study telomere length, which in vertebrates are repetitive hexameric sequences (TTAGGG) located at the distal end of chromosomes. Telomeres are necessary at chromosome ends to prevent DNA-damage responses as well as genome instability. To this day, the Q-FISH method continues to be utilized in the field of telomere research.

Telomere-binding proteins function to bind telomeric DNA in various species. In particular, telomere-binding protein refers to TTAGGG repeat binding factor-1 (TERF1) and TTAGGG repeat binding factor-2 (TERF2). Telomere sequences in humans are composed of TTAGGG sequences which provide protection and replication of chromosome ends to prevent degradation. Telomere-binding proteins can generate a T-loop to protect chromosome ends. TRFs are double-stranded proteins which are known to induce bending, looping, and pairing of DNA which aids in the formation of T-loops. They directly bind to TTAGGG repeat sequence in the DNA. There are also subtelomeric regions present for regulation. However, in humans, there are six subunits forming a complex known as shelterin.

<span class="mw-page-title-main">Mega-telomere</span>

A mega-telomere, is an extremely long telomere sequence that sits on the end of chromosomes and prevents the loss of genetic information during cell replication. Like regular telomeres, mega-telomeres are made of a repetitive sequence of DNA and associated proteins, and are located on the ends of chromosomes. However, mega-telomeres are substantially longer than regular telomeres, ranging in size from 50 kilobases to several megabases.

<span class="mw-page-title-main">Peter M. Lansdorp</span> Dutch medical researcher

Peter Michael Lansdorp is recognized for his contributions in the fields of hematology, medical genetics and cancer research. He has made significant contributions to the understanding of genome instability, particularly in relation to aging and cancer. His research has focused on the biology of blood-forming stem cells, telomeres and genome analysis. He is also known for developing techniques including single cell Strand-seq and fluorescence in situ hybridization (FISH) techniques such as Q-FISH and flow FISH.

References

  1. 1 2 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. 1 2 3 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.
  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. 1 2 Lansdorp, 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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