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Keeping check of an In-situ hybridisation experiment in general and making good - (Sep/17/2009 )

Hi,

First some random (or not so random) thoughts:

From my undergraduate degree in biochemistry I learned that to check yield/efficiency etc. of each step in a long protocol (i.e. purification) is golden.

Now In my graduate research Im facing whole-mount In-situ hybridisation. Its a long protocol and it seems rather "black-boxy" to me. See, you only get to kno wheter the experment worked or not after a weeks worth of work. And then you have all the parameters to play with at once without knowing which "failed"! No tweaking of individual incubation times/temperatures or such with immediate feedback as in biochemistry.

As such I will know refere WMISH as the "black-box" experiement :D


So to the work at hand, with this background in consideration:


I want to make good hapten ssRNA probes (about 1kb, from PCR products of plasmids), but how do I know the probes are good?

I dont want to have to go through several weeks of ISH with probes that dont function to begin with!!!


cheers,

mordiano :) :)

-mordiano-

You could try dot-blotting or other hybridisation technique (northern blot) to test if the probe will bind to the RNA, and under what conditions.

-bob1-

Thanks for the comment,

After checking around the department dot/blot seems to be the standard way to do it...

Nevertheless, what cought my attention was an approach that involves doing a regular EtBr agarose gel with the 1. starting DNA, 2. DNA+Synthesis (adding a new RNA band), and 3. DNA+Synthesis+DNAse (removing the DNA band).

The gel should be run at high voltage, reducing time for rnases to work, and with as clean as possible reagents.

The problem i see from this are the famous RNAse-ses, but then again everyone seems to have diffrent opinions on thoose..


regards,

mordiano

-mordiano-

Why not label the RNA probe with a FITC -dUTP , and run it through a column to remove any unlabelled nucleotides? If your PCR worked correctly, the pellet should be bright green. You could always run an RNA gel to double-check the size too.

-Katy-

Katy,

That would make my ISH flourescent ISH right?

..or do you mean to combine both the hapten (Ab-binder) and the FITC?

regards,

mordiano

PS. And that woul'd be seen by the naked eye?

-mordiano-