A simple and rapid fluorescence in situ hybridization microwave protocol for reliable dicentric chromosome analysis

Abstract

Fluorescence in situhybridization (FISH) is an extremely effective and sensitive approach to analyzing chromosome aberrations. Until recently, this procedure has taken multiple days to complete. The introduction of telomeric and centromeric peptide nucleic acid (PNA) probes has reduced the procedure's duration to several hours, but the protocols still call for a high temperature (80–90°C) step followed by 1–3 h of hybridization. The newest method to speed up the FISH protocol is the use of a microwave to shorten the heating element to less than a minute; however this protocol still calls for a 1-h hybridization period. We have utilized PNA centromere/telomere probes in conjunction with a microwave oven to show telomere and centromere staining in as little as 30 s. We have optimized the hybridization conditions to increase the sensitivity and effectiveness of the new protocol and can effectively stain chromosomes in 2 min and 30 s of incubation. We have found that our new approach to FISH produces extremely clear and distinct signals. Radiation-induced dicentric formation in mouse and human fibroblast cells was analyzed by two individual scorers and the observed dicentrics matched very well.

Rapid detection of t(15;17)(q24;q21) in acute promyelocytic leukaemia by microwave‐assisted fluorescence in situ hybridization

“…Microwave oven use has become a daily routine in diagnostic pathology of solid tumours, particularly in immunohistochemistry, whereas MW‐FISH on tissue sections is reserved for pathology archive samples, where the quality of signals is poor . MW‐FISH has already been applied in many sample types, with a large variety of probes, different in size and target . However, there are very little data concerning the application of MW‐FISH in cytogenetics, on metaphase cells or interphase nuclei, and they are about constitutional aberration detection .…”

“…MW‐FISH has already been applied in many sample types, with a large variety of probes, different in size and target . However, there are very little data concerning the application of MW‐FISH in cytogenetics, on metaphase cells or interphase nuclei, and they are about constitutional aberration detection . This is the first report concerning the application of MW‐FISH in the genetic diagnosis of hematologic malignancies.…”

Direct DNA and PNA probe binding to telomeric regions without classical in situ hybridization

“…B70 cells were cultured in Minimum Essential Medium Alpha media (Hyclone, ThermoFisher, Waltham, MA) with 15% FBS (Sigma, St. Louis, MO) antibiotics (Anti-Anti, Invitrogen, Indianapolis, IN) [5]. …”

Ascorbic Acid 2-Glucoside Pretreatment Protects Cells from Ionizing Radiation, UVC, and Short Wavelength of UVB

“…Just after irradiation, G2-PCC was induced by adding 50 nM calyculin [34]. Thirty minutes after incubation, cells were harvested and chromosome spreads were prepared as a standard protocol [35,36]. G2-PCC was analyzed with the frequency of chromatid type aberrations (chromatid types of gap, break and exchange) per cell.…”

“…Furthermore, 12 h after exposure, 0.2 µg/mL of colcemid (Invitrogen) was added to cells and allowed to incubate for an additional 6 h. Cells were harvested and then suspended in 4 mL of 75 mM KCl solution and placed in a 37 • C water bath for 20 min. A fixative solution of 3:1 methanol to acetic acid was added to the samples according to the standard protocol [35,36]. Giemsa stained metaphase chromosome images were taken using a Zeiss Axioplan microscope (Zeiss) equipped with Q-imaging Aqua CCD camera and Q-capture Pro software (QImaging, Surrey, BC, Canada).…”