MiRNA isolation

[mohamed samir]

Hi everybody. Iam doing isolation of miRNA from tracheal tissue of infected mice. What is bothering me really that I got surprised when I end up with low RNA yield of the sample. I red the protocol carefully and found nothing wrong with my work. Have any one knowledge what is the difference between chlroform and the chloroform /isoamyl alcohol mix. I mean the protocol stated that you have to be sure that the chloroform is not mixed with iso amyl alcohol. But I red that the iso amyl alcohol reduces the RNAase so i suppose that it will be beneficial for the isolation.

I would appreciate it if any one help me.

[CharonY]

The presence of isoamylalcohol is usually for stabilization of the choloroform and reduction of foaming. There are a lot of different protocols however, and if it explicitly states that it should not be used, there may be a reason. Other than that chances are that you got RNAses into your extraction, need to work a bit faster, keep the work area cleaner etc.

 

With multi-step extractions and especially with the low stability of RNA it usually takes a bit of practice to get consistently good results. If the yield is consistently low, however, it is time for running controls for troubleshooting.