Immediately frozen liver with dry ice, I cut a piece of 100mg, I add 1 ml of Trizol reagent, homogenized with homogenizer power and proceed to phase separation, precipitation and RNA wash following the supplier's instructions, and resuspend the RNA pellet in RNase-free water, but when I make a gel to check the integrity of the RNA, it is partially degraded. My question is how do I extract liver RNA without degrading it?
Hi Celeste
Liver does contain majority of RNAase, better to keep your samples first in RNAlater solution then try the same. Rather using RNAase free water use either RNA storage solution or Tris pH 7.2-8 for storage of RNA.
All the spin should done in 4 degree and all reaction (Chloroform or isoprop) in RT for specific period time.After isoprop treatment it is best to keep the samples in -80 for over night for better yield.
Hop will help
Hi Celeste
Liver does contain majority of RNAase, better to keep your samples first in RNAlater solution then try the same. Rather using RNAase free water use either RNA storage solution or Tris pH 7.2-8 for storage of RNA.
All the spin should done in 4 degree and all reaction (Chloroform or isoprop) in RT for specific period time.After isoprop treatment it is best to keep the samples in -80 for over night for better yield.
Hop will help
Hi Celeste,
RNA extraction is very sensitive to RNAses. To limit their action:
- as Abhijit said, centrifugation should be performed at 4°C,
- unless other mention from the manufacturer for specific steps, the whole process should be performed on ice and not at room temperature,
- it's important to work in a RNAse free area: clean your bench with bleach, use RNAse free pipettes, tips, eppendorfs, wear gloves at all time (that you didn't touch with you bare hands, at least the outside of them) etc.
RNA extraction really require an environment in which RNAse won't be active.
Good luck,
Audrey
You may change the water for RNA dissolving (take new one) or add RNAse inhibitor (like RNasine) in water before resuspend the RNA pellet if you not sure in "RNase-free". For all reactions with RNA it is better to use DEPC-treated water (autoclaved during 3 h, 1,5-2 ext atm). Use the voltage during the phoresis as low as possible (e.g. 100V). Try to avoid RNA overload in a gel well (you should make the phoresis with some serial 10-fold dilutions of the RNA sample).
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