InSitupedia
This method is mainly applied to detect DNA sequences that are not easily detected by conventional ISH.
It uses nonisotopic indirect method with digoxigenin.
This method allows increase of the sensitivity of ISH signals by amplification of detecting systems or amplification of the DNA or RNA targets with In Situ Polymerase Chain Reaction.
Combines the high sensitivity of PCR with the anatomical localization by ISH.
PCR can amplify low copy DNA sequences to high levels but cannot correlate results with morphological features.
ISH localizes genes sequences at the cellular level but it requires at least 20 copies of the mRNA of interest in a cell to make the signals visible.
The combination of PCR with ISH can amplify specific DNA or RNA sequences inside single cells and increase the copy numbers to levels readily detectable by ISH methods
Main approaches:
Direct – a labeled nucleotide is corporate into PCR products
Indirect – ISH is performed after in situ amplification using labeled oligonucleotide probe
PCR ISH is used for:
-
Detect low copy number of viral genes, HIV and hepatitis C.
-
Leading to significant discoveries about vital infection diseases.
Protocol: a fast and simple in situ PCR method for localising gene expression in plant tissue
In Situ Reverse Transcription allows to detect gene expression in cell preparations and frozen sections, successful amplification of mRNA of hormone, receptor, oncogenes.
For mRNA analyses the use of RT-PCR allows to increase the hybridization signals from 2 to 20.
Can detect very low levels of nucleic acids in tissues by taking advantage of the powerful amplification capacity of PCR.
In situ reverse transcription: the magic of strength and anonymity
