I have some intergenic small RNA candidates in prokaryotes which I want to validate.
Insitu hybridization using LNA, Q-PCR using taqman probes for specific small RNAs.
Hi Harry
In addition to the approaches listed by Dasaradhi above...check also the possibility of using hari-pin primer based qRT-PCR.........but i feel Northern blotting is still very much ...like a gold standard...for small RNA analysis......
bye
We routinely use Real-Time PCR with Taqman probe in our lab, to verify induction or repression of microRNA in various condition. At the biological level, to verify the effect of microRNA we use microRNA mimics and inhibitors. We also use RT-PCR to verify if the microRNA mimics or inhibitors worked properly.
Hi,
I used qRT-PCR combined to the use of microarrays to validate CUT .
Thanks everybody for the suggestions. Even I am thinking of proceeding with qRT-PCR qith Taqman probe, but I am not sure whether this system is worked out in prokaryotes, as I was not able to find any reference.
Other doubt I have- in the hairpin primers, the stem loop part is designed and commercially available for eukaryotes, but what if I want to design for prokaryotes, any non-specific sequence is enough with similar melting behaviour, or some other strategy is involved for the primer design
go for Northern blotting, with strand specific probes. This is the best to check expression and size. Once validated, you can go on with regulatory studies using qRT-PCR, but Northern blotting is the reference method to validate.
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