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Multiplex FISH and Spectral Karyotyping
Multicolor fluorescence in situ hybridization (mFISH) assays are indispensable for a precise description of complex chromosomal rearrangements and marker chromosomes. Routine application of such techniques on human chromosomes started in 1996 with the simultaneous use of all 24 human whole-chromosome painting (WCP) probes in multiplex-FISH (M-FISH) and spectral karyotyping (SKY). Here we present a review on the available mFISH approaches using WCP probes and describe a basic protocol for M-FISH and SKY. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Two- to Three-Color FISH
Two-color FISH is normally done to apply simultaneously a specific and a control probe. Three-color FISH can be done also as a combination of control and specific probes; however, also three specific target regions may be addressed at the same time. Here the application of commercially available two- and three-color FISH probes is described, and applications in diagnostics and research are discussed. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

FISH on Sperm, Spermatocytes and Oocytes
It is well known that chromosome in situ hybridization allows the unequivocal identification of targeted human somatic chromosomes. Different fluorescence in situ hybridization (FISH) techniques have been developed throughout the years, and following the mitotic studies, meiotic analyses have been performed using these different techniques. The application of FISH protocols on meiotic cells requires adaptation of standard protocols to the particularities of these cells. Specific sample fixation is usually required, and in some cases samples need to go through particular pretreatments to guarantee a successful FISH. The app...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Characterization of Archived Formalin-Fixed/Paraffin-Embedded or Cryofixed Tissue, Including Nucleus Extraction
Interphase cytogenetics using archival tissue samples is a straightforward approach to obtain“cytogenetic information” from nuclei of solid tissue samples. It is the major tool to investigate specific numerical chromosomal aberrations, chromosomal translocations, amplification of oncogenes, or deletion of tumor suppressor genes in archival tissue samples on a single-cell level, as no metaphase spreads can be obtained, obviously. Here a collection of protocols for archival sectioned and mounted material as well as extracted nuclei (from formalin-fixed/paraffin-embedded or cryofixed tissues) is presented. (Source...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Characterization of Mosaicism in Different Easy-to-Acquire Body Tissues Such As Buccal Smears, Skin Abrasions, Hair Root Cells, or Urine
Mosaic karyotypes are present in at least 0.3 to 1 % of clinical cases analyzed by banding cytogenetics. It is well known that the pattern of mosaicism can be extremely variant in different tissue types of the same patient. However, normally in maximum two different tissues of a child or an adult are cytogenetically studied, i.e., peripheral blood and skin fibroblasts. Here preparation protocols for four easily acquirable further tissues are presented. The resulting preparations of interphase nuclei can be used in routine interphase FISH applications. Here it is summarized how to treat buccal smears, skin abrasions,...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Tumorcytogenetic Diagnostics and Research on Blood and Bone Marrow Smears or Effusions
Blood smears, bone marrow smears, and effusions can be very helpful for quick molecular cytogenetic diagnostics in leukemia, lymphoma, or even solid tumors. Such samples can be studied by suited interphase FISH probes or probe sets for diagnostics, prognostics, therapeutic decisions, follow-up and control of effects of medication, or success of bone marrow transplantation. Here two protocols are provided on how to prepare smears or cell suspension in methanol/acetic acid (3:1) for subsequent FISH analyses. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

FISH in Uncultivated Amniocytes
In prenatal diagnostics, interphase FISH (iFISH) may be applied for the detection of certain numeric chromosomal aneuploidies. iFISH in uncultivated amniocytes can be performed from 12 weeks of gestation to the third trimester. Alternatively, the described aneuploidy screening set here is also suitable on short-term or uncultivated chorionic villus sampling (CVS) without metaphases as a fast screening test for the most common aneuploidies. Also this test helps to distinguish between culture-induced or real fetal triploidy or tetraploidy (mosaicism), which is found from time to time in the parallel cell culture; in this set...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Application of FISH to Previously GTG-Banded and/or Embedded Cytogenetic Slides
GTG-banded slides are often considered to be worthless for further molecular cytogenetic analyses. In this chapter we describe a simple protocol on how to reactivate such GTG-banded slides for further use in molecular cytogenetics. The slides can be previously Eukitt® or“Canada Balsam” embedded or previously not covered with a coverslip at all. With this possibility at hand, archival material of rare cytogenetic aberrations as well as more profound studies on actual cases with limited access to material can be performed. (Source: Springer protocols feed by Cell Biology)
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Pre- and Postnatal Diagnostics and Research on Peripheral Blood, Bone Marrow, Chorion, Amniocytes, and Fibroblasts
Peripheral blood, amniocytic fluid, chorion, and fibroblasts are the most frequently used tissues for chromosome studies. All four of them are relatively easy to obtain and can simply be brought into short-term culture, and metaphase spreads can be prepared within rather short time. Such metaphase spreads, as well as the huge amounts of previously superfluous interphase nuclei in cytogenetic preparations, are a material very well suited for FISH analyses. All kinds of FISH probes can be used to analyze metaphase spreads, while the interphase nuclei actually can be analyzed for routine purposes only by satellite and locus-s...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

The Standard FISH Procedure
Molecular cytogenetics originally comprised of two basic approaches: fluorescence in situ hybridization (FISH) and primed in situ hybridization (PRINS). Nowadays FISH is the one routine approach still used in research and routine molecular cytogenetics field. Here the basic protocol in how to do FISH using commercial and/or homemade DNA probes is described. For a protocol for FISH on tissue sections, please refer to chapter by Thomas Liehr“ Characterization of Archived Formalin-Fixed/Paraffin-Embedded or Cryofixed Tissue, Including Nucleus Extraction .” Besides direct-labeled probes, also ind...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

The Replicative Detargeting FISH (ReD-FISH) Technique in Studies of Telomere Replication
Based on the chromosome orientation-FISH (CO-FISH) procedure, the replicative detargeting FISH (ReD-FISH) was developed as a unique tool to study the replicative patterns of telomeres located on individual chromosomal arms. This method is also suited for examination of telomeres of species belonging to different classes of animals, for which well-proliferated cell cultures can be established and maintained. ReD-FISH is based on pulsed inclusion of labeled brominated nucleotides in replicating DNA, destruction of regions with brominated analogs, and standard FISH with single-strand-specific probe/probes. (Source: Springer p...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

RNA Imaging in Living Cells
Insight into the dynamics of RNA biosynthesis, processing, and cellular activity is highly valuable because it will deepen our understanding of cell physiology and help explain how mRNA-misregulation contributes to the development of many diseases. To date, the study of mRNA inside cells is a challenge; many analytical approaches focus only on quantifying expression levels of transcripts and are not capable of reporting their intracellular locations, which have emerged as a critical determinant of RNA function. Similarly, techniques capable of probing RNA localization merely offer snapshots in fixed cells. Herein, we descr...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Telomere Length Measurement by FISH
Telomere length influences numerous cellular processes such as senescence, carcinogenesis, and aging. Quantitative FISH (Q-FISH) is a comprehensive method that allows measuring of individual chromosome telomere length in single cell with the resolution of 200 base pairs. The method is based on the use of a peptide nucleic acid (PNA) telomere oligonucleotide probe and appropriate digital image software for capture and quantification of fluorescence signals. The length of telomere is directly related to its integrated fluorescence intensity, as PNA probes are assumed to hybridize quantitatively to telomeric repeats. For the ...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

One-Day Quick FISH
The classical FISH approach requires overnight incubation for proper hybridization result. Tissue morphological features are varying due to aggressive pretreatment and high temperatures applied. To increase the speed of the molecular cytogenetic finding and eliminate some of the technical limitations, an alternative one-day FISH method was recently introduced. This procedure allows the completion and evaluation of a FISH reaction within one day by the reduction of the hybridization time to 60–120 min. Moreover, the low denaturation temperature significantly contributes to better tissue and cell morphology of the...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news

Formamide-Free Fluorescence In Situ Hybridization (FISH)
Formamide is an ionising solvent which is widely used in molecular biology research for its thermodynamic effects on the DNA double-helix stability. In fluorescence in situ hybridization (FISH), the addition of formamide to aqueous buffers solutions of DNA enables key procedural steps—such as the prehybridization denaturation, the reannealing step and the post-hybridization stringency washes—to be carried out at lower, less harsh temperatures without compromising the overall efficiency and specificity of the hybridization. However, formamide is toxic and a potential teratogen, and its use in research laboratori...
Source: Springer protocols feed by Cell Biology - October 20, 2016 Category: Cytology Source Type: news