RNA extraction protocol for total RNA - TAU：总RNA提取的RNA头协议.doc

RNA extraction protocol for total RNA This protocol uses Trizol (also known as TRI REAGENT) for the isolation of total RNA. Trizol is a mixture of guanidine thioacyanate and phenol, which effectively dissolves DNA, RNA and protein on homogenization or lysis of tissue sample. After adding chloroform and centrifuging, the mixture separates into 3 phases with the upper clear aqueous phase containing the RNA. The next steps in the extraction are washes and precipitation of the RNA. The first part of the protocol – from the homogenized tissue in Trizol to the point of an RNA pellet in 75% ethanol, takes less than 1 hour. The RNA can then be stored for long periods of time, at -200c. The same protocol can be used for RNA extraction from cell cultures. For further use of the RNA for expression analysis, it is highly recommended to treat the sample with DNase, an enzyme that digests DNA. This procedure is very effective for isolating RNA molecules of all types from 0.1 to 15kb in length. However, there are commercial kits that enable simple RNA extractions, usually using a column that binds the RNA, and also include the DNase treatment in them. Moreover, inherent to methods that use phenol-chloroform for RNA isolation and cleanups is a certain loss of total RNA. This varies in percentage depending on the sample size (the larger the amount of total RNA, the smaller the relative loss). I therefore recommend using this protocol for RNA isolations from large number of cells. However, once laser microbeam dissected RNA extraction from cells will be considered, I strongly recommend using a different method, preferably a suitable kit for such extractions. This modification could be considered also in the case of RNA extraction from sensory epithelia. Reagents required: It is highly recommended to keep separate stocks as well as pipettes and tubes that will be used for RNA. Always work in an RNase free environment, after cleaning pipettes and table with RNase away or a similar co