miRNA in situ hybridization - (Nov/26/2007 )

I want to try miRNA in situ hybridization . I wonder what kind of probe should i use?

LNA may be first choice,but I'm afraid it's too expensive.

then ,should I use synthetic RNA or DNA probe? optionally, some residue modified?


Anybody tried any of them? Thank you in advance!

I will be trying the LNA probes for the first time later this week. I can let you know how it works if you like - just send me a PM so I don't forget.

I have tested LNA probe for in situ hybridication and found it did show signal, while normal DNA oligo did not.
However, I still worry about cross hybridization since in Northern hybridization using LNA probe, I noticed strong hybridication signal come from high molecuar (HMW) RNA, just as illustrated in Fig. 1 of the first paper using LNA probe for miRNA (Nucleic Acids Res. 2004 Dec 14;32(22):e175), and it was rare to found such HMW hybridization sigal when using normal DNA oligo as probe in Northern hybridization...

any idea for it?

Well since the amount of precursor molecule is usually low for each cell, maybe you see the HMW just because the LNA probes have increased stability once bound to the target? i.e. LNA will bind 90% of available HMW wherease the normal DNA will bind a far lower amount??

Well, miRNA probe could not bind to miRNA target, therefore, HMW signal seem derived from unspecific binding.
The caution is that such possilbe unspecific signals are stronger than miRNA's, and they might also exist in the work of in situ...

i used LNA , 5' labeled probes for the in situ on mouse tissues ---worked great. probes were 100% complimentary to mature miRNA. I didnot see any nonspecific binding.