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. It´s 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


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

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.

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

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,

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?¿