By Marilena Aquino do Muro, Ralph Rapley
CABI Bioscience united kingdom Centre, Surrey, united kingdom. offers the foundations for gene probe layout, labeling, detection, aim layout, and hybridization stipulations including special protocols, sensible tricks, and suggestions. For researchers. define structure.
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Extra resources for Gene Probes, Principles and Protocols
The INK4A gene is localized in a chromosomal area found to be frequently altered in bladder transitional cell carcinomas (31). This, in addition to the functional and genetic evidence that supports an important role for INK4A From: Methods in Molecular Biology, vol. 179: Gene Probes: Principles and Protocols Edited by: M. Aquino de Muro and R. , Totowa, NJ 43 44 Orlow and Cordon-Cardo Fig. 1. Genomic organization of the INK4A and INK4B gene locus. as a tumor suppressor gene in a variety of tumors prompted several groups to study the relevance of the INK4A/B locus in bladder tumors.
5 µL of each probe (and competitor probe if required, see Note 5) at the working concentration of 50 ng/µL, and mix carefully with pipet tip (avoid touching surface of slide with pipet tip as this will disturb attached cells). Place the slide in the 50 mL tube containing the moistened towel. Screw on cap and place horizontally into hybridization oven at 46°C for 1–2 h. 01%) Following hybridization, rinse wells immediately with 48°C wash buffer into the hybridization tube, using a pipet. Carefully remove slide from the hybridization tube, place into wash buffer tube, and hold at 48°C for 10–15 min.
Relative amounts of a given CKI gene (INK4A or INK4B) present in each sample are determined by comparing gene-specific hybridization signals with those obtained using the control probe, and are expressed as ratios (target-band signal)/ (control-band signal). Some examples are shown schematically in Fig. 3. If the tumor tissues contained originally approx 70% of tumor cells, samples with <30% of the control signal are considered homozygously deleted; and those presenting 31–65% as hemizygously deleted for the INK4A/B specific genes.