In Situ Hybridization ISH Flashcards
What is hybridization?
The process of combining two complimentary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing (probe binds to its target in 20 min to 18 hours)
What is a probe?
A single-stranded sequence of DNA or RNA used to search for its complimentary sequence in a sample genome
What is a (positive control) DNA probe?
Human placental DNA, poly T, or other probe used to monitor availibility of tissue nucleic acid
What is a negative control probe?
Unmodified plasmid, unrelated probe, sense probe, or other probe used to monitor specificity of hybridization and detection
What is a positive control probe?
A control tissue positive for the probe of interest
What is pretreatment?
The process of tissue permeableization which allows the probe to reach teh target
Can be done with heat, Proteinase K, Triton X-100, SDS, or other detergents
What is stringency?
A term in hybridization indicating the homology or “matchiness” between the probe and the nucleic acid
What are the methods for pretreatment of tissue?
Heat mediated Enzyme digestion (be careful not to over-digest)
What causes weak staining?
over or underfixation incubation too short low RNA expression RNase contamination overdevelopment of substrate left out a critical reagent such as digestion or retrieval solution incorrect hybridization temperature
What causes weak/no staining?
over or underfixation incubation too short low RNA expression RNase contamination overdevelopment of substrate left out a critical reagent such as digestion or retrieval solution incorrect hybridization temperature
What causes background staining?
varriable fixation overdeveloped substrate inadequate tissue rinsing incomplete tissue deparaffinization damaged or necrotic tissue sections dried out during hybridization
RNase
digests RNA, wear gloves and have sterile glassware so you don’t mess up your ISH
Rehydration
The process of removing paraffin from the tissue by immersion in xylene, gradded, alcohols, and ending in water
In-Situ Hybridization (ISH)
Binding complimentary single strand DNA or RNA to a target in preserved tissue to localize mRNA in cells/tissue
