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Detection of ribosomal DNA sites by Fluorescence in situ hybridisation (FISH) (CAT#: STEM-MB-1197-WXH)

Introduction

Ribosomal DNA (rDNA) codes for the rRNAs used in the production of ribosomes, the sites of cellular protein synthesis. In most eukaryotes, ribosomal DNA consists of tandemly repeated arrays of four or five genes located at the nucleolus organizer region (NOR) of one or more chromosomes.




Principle

FISH uses fluorescent probes with complementary base sequences to locate the presence or absence of specific portions of DNA on chromosomes. The probe and target DNA must be denatured with heat or chemicals to break hydrogen bonds in the DNA and to allow hybridisation to occur once the two samples are mixed. The fluorescent probes form new hydrogen bonds with their complementary base pairs on the DNA, and these can then be detected via microscopy.

Applications

Detect and localize the presence or absence of specific DNA sequences on chromosomes.
Detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples.

Procedure

1. Sample preparation
2. Co-denaturation and hybridization
3. Probe detection
4. Wash off of unbound probe
5. Analysis by flow cytometer/fluorescence microscopy

Materials

• Flow cytometer
• Fluorescence microscopy
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