miRNA extraction from primary tumor samples - (Oct/31/2006 )
I was doing a miRNA extraction from primary tumor samples (using miRNeasy mini kit) and the total RNA yield was quite low. I think the problem lies in the homogenization step. I have to homogenize the samples manually and to be honest found this very difficult. I still had clearly visible pieces of tissue in the homogenate.
I was wondering can anyone suggest any good manual methods for disruption and homogenization of the tumor samples as i do not have access to any homogenizers. I literally placed the tissue (about 50mg) into the QIAzol lysis reagent (700ul) and tried to homogenize using the pipette. This obviously was not effective. Can anyone suggest any other possible methods?
What you need to do is find a tissue homogenizer that uses a metal beads, like a bead beater, Geno/Grinder, etc.
You can also try pulverizing the tissues in liquid nitrogen then adding a Triazol or your QIAzol reagent.
I using a pestle and mortar and grind the tissue frozen in liquid nitrogen. Snap freeze your sample and try to cut it into smaller pieces with a skalpel before grinding. add nitrogen frequently so that the sample does not thaw ( i know this is really difficult but try
Other people in my department use a glass homogenizator and autoclaved sea sand for disruption, but i have never tried that.
all the best,