RNA Isolation from Breat Normal tissue - (Feb/27/2006 )
I'm trying to isolate RNA from Breast Normal tissue but the yield is very low (the concentration is low and the total amount is low too), so I can't contimue working with the RNA.
I'm using TRI-REAGENT protocol (it works as Trizol prep).
I used to isolate RNA from other tissues in the same way, but I had no problems than and I got high yieled. the problem is only with breast normal tissue, maybe because it contains high precent of fatty tissue.
Does anybody has any experience with this?
hi there is a section in tri protocol
OPTIONAL: An additional isolation step may be required for samples with high content
of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and
tuberous parts of plants. Following homogenization, remove insoluble material from the
homogenate by centrifugation at 12,000 ×××× g for 10 minutes at 2 to 8°C. The resulting pellet
contains extracellular membranes, polysaccharides, and high molecular weight DNA,
while the supernatant contains RNA. In samples from fat tissue, an excess of fat collects as
a top layer which should be removed. In each case, transfer the cleared homogenate
solution to a fresh tube and proceed with chloroform addition and phase separation as
Thanks for the information.
Just one more questioun- do you know what is the expected yield that I suppose to get from 500mg tissue?
half a kg of tissue???
maybe it would be better to split the tissue in less weighted samples... for 2 reasons at least :
1 : if you fail one exp, stock still be available
2 : cell lysis is better on small volumes
For total yield, i don't know right value.
Did you download the manual i've attached?
There is a section in which it's told that 1mg of rat liver yields 16 +/- 4 µg RNA
3 mg liver yields 23 +/- 5 µg RNA
5mg liver yields 38 +/- 2 µg RNA
10mg liveryields 77 +/- 3 µg RNA
Note that the yiled per mg decreases with the increase mass of starting sample.
So assuming fat is more present in breast tissue, and if you divide your sample in pieces of 50mg, you will probably retain 200 to 250µg per assay of 50mg...
just an idea.But i still believe that you may do a test with 1/10 (and better if on this 1/10 you split this in 5 to 10 samples too) and get the idea for global exp.