Hi Hao,

to my knowledge there is no "general" principle how to determine your hybridization temperature based on the lenght of your riboprobe - the temperature usually is empirically optimized.

However here are some rules:

When you have really short probes you use lower temperatures (maybe 42°C and lower).

For longer probes you use higher temperatures - I usually started with 52-55°C for probes from 200 bp to probes more than 1 kb in size - this worked usually well.

These are recommendations for the detection of RNA with RNA probes. If you want to detect RNA with DNA probes you should use lower temperatures due to the fact that the hybrid molecules tent to be less stable (e.g. around 45°C).

What is also pretty important is the salt concentration in your hybridization buffer because this strongly influence the stringency of your puffer (lower salt and higher temperature usually means you have a higher stringency.

The stringency of you washing buffer (and the washing temperature) can alos greatly influence the outcome of you ISH. UUsually you start with lower stringent washing conditions and increase stringency during washing. You might find these publication regarding ISH techniques helpful - its for FISH but I think the principles are also applicable for otherr kinds of ISH:

Article A technical review and guide to RNA fluorescence in situ hybridization

KR Dörthe

Dörthe, thank you very much for your reply in detail, which helps me a lot. Hao

Dörthe Andrea Kesper

Dear Dr. Kesper

Can you help me?

Tm probe temperature and Hybridization temperature is related to each other for DNA FISH by oligonucleotide probe?

What temperature should we roughly consider for each?

I would be grateful if you read my question in the link below.

https://www.researchgate.net/post/How_do_I_design_FISH_probe_for_sperm

Hi Marjan,

I am not a specialist for DNA FISH, I have done it only once and this was quite a while ago.

However, I will see what I can find regarding this protocol and if I am able to give you some useful tips but I can't guarantee this. I will come back to you later.

KR Dörthe