In situ PCR

David Fogarty onxfoxxd at lgdx02
Mon Jul 1 08:47:09 EST 1996


To anyone performing in situ PCR on histological slices:

We have done a number of experiments to detect RNA encoding various 
receptors in brain slices (both paraffin embedded and cryostat prepared 
slices). Initially we used the direct method with fluorescent labelled 
nucleotides but background was always high despite high stringency 
washings.  

Now we plan using the more reliable method of doing ish on pcr amplified 
sections.  My question is as follows:

Which type of labelling strategy for the specific internal probe is best?

Options provided by many commercial companies include:
5' or 3' labelling of oligos with:
biotin
fluorescein
digoxigenin
alkaline phosphatase
and
internal labelling with the same reporter molecules.

What, apart from the very obvious reasons (like economic), are the 
advantages of one reporter molecule over the others?
What is the advantage of 3' labelling over 5' labelling?

Comments from anyone working with this technique would be appreciated.

Thanks,

David :)






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