hey guys , Would you like to share your RNA extraction protocol?
I've been in different lab and find there are lot different between the protocol.
Usually the all step performed on the ice
first step is add trizol, mixed well and stand for few mins,
add chroloform, votex and stand for few mins,
centrifuge and trasfer supernant on new tube
add isoalchol and revert, store at -80℃ >30mins or just stand on ice
centrifuge and remove supernant
wash with 80%alcohol for two times
dissolve in RNAase free water.
what's ur centrifuge speed and time? do you guys store at -80℃?
I think there is no best protocol for RNA isolation. It depends on what type of organism, tissue, cell you work with (and also on the type of RNA you want to isolate). The basic protocol follows the steps you mentioned. Precipitation in alcohol (isopropanol or ethanol) is usually followed by a high speed centrifugation step (I used to do 14000 rpm for 30 min at 4°C in a bench-top centrifuge). For washing I used 70% ethanol and centrifuged for 10 min (same speed and temperature parameters as before). Storage of RNA at -80°C is highly recommended.
Regards, Christian
your protocol looks ideal.
In my case I found the precipitation at -80 degrees is better than on ice. Precipitation at -80degrees reduces RNA degradation if there is any possible RNase contamination and also you can leave the samples for longer time, even over night.
As Scheckhuber said, I also did centrifugation at maximum speed for 30mins at 4degrees.
Better to store isolated RNA in small aliquots at -80degrees.
The most important step is collection of clean aqueous phase after the very first centrifugation step - sacrifice yield over purity. If necessary, add extra chloroform and respin, collect clean aqueous phase for alcohol precipitation.
If the above step is done carefully, all RNase is inactivated and eliminated. RNA in the absence of RNase is quite stable, you can use any temp. In the range of 4C - -80C that suits your work flow or makes you feel comfortable (unless you have very little RNA, in which case add glycogen to help precipitate RNA),.
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