Hi everyone!
I use HaCaT cells and i transfect them with siRNA. After 48 h I extract proteins with Laemmli and RNA with trizol. I proceed with western blot to check proteins levels (and they are good) and i proceed with rtPCR and qPCR to see if the gene is overexpressed.
The problem is that with the same tratment i have sometimes:
- low expression of this gene
- really high expression
Why there is this variability?
I don't understand what i do differently than the other times when i do the same experiment.
Can RNA extraction be crucial on ubiquitin gene expression?
There are several factors to keep in mind in this kind of experiments:
1. RNA uses to degradate very easily. I recommend you to perform the complementary DNA assay the same day of the RNA extraction and the qPCR next day if possible.
2. RNA expression in the cell is highly variable. It depends on passage, confluency, medium refresh... I know sometimes is hard, but you should obtain your samples always in the same conditions.
3. Aliquot your treatments. Thaw and freeze cycles reduce the effectivity of most drugs. Try to make tiny aliquots and freeze no more than 2 times.
4. Make more replies to assure the result!
I hope these advice could help you in some way.
To perform the ubiquitin western blot, a specific protease inhibitor should be added, N-ethylmaleimide.
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