RNA Isolation by Trizol Method
This RNA isolation by Trizol method protocol uses Trizol which is also known as TRI REAGENT for the isolation of total RNA. Trizol is a mixture of guanidine thiocyanate 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, inter phase containing cell debris and lower is organic phase having protein, lipids. The next steps in the extraction are washes and precipitation of the RNA. The first part of the protocol – from the homogenized tissue inTrizol 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 -20°C. 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 15 kb 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).
Requirements for RNA isolation by Trizol method
The followings are the requirements for this test procedure-
- 1.5 mL Eppendorf tubes
- RNAse free water
- Micro pipette and tips
- Test specimen
Procedure of RNA isolation by Trizol method
- Add TRIZOL reagent to the cells (sample, 100 µl, and Trizol, 300 µl ) and incubate at room temperature for 5 minutes.
- Transfer the cells lysate to a 1.5 mL centrifuge tube and add 0.2 mL of chloroform.
- Mix it thoroughly and incubate at room temperature for 5 minutes.
- Centrifuge the mixture to centrifugation at 12000× g for 15 minutes at 4 °C.
- Transfer the aqueous phase containing total RNA to a fresh tube and precipitate the RNA by adding 0.5 ml of isopropanol followed by incubation at room temperature for 10 minutes.
- Centrifuge the precipitate at 12000× g for 10 minutes at 4 °C.
- Discard the supernatant and air-dry the RNA pellet for 10 minutes and resuspend in 20 µl of RNAse free water.
- Perform agarose gel electrophoresis to check the integrity of the RNA.
Observation of step RNA isolation by Trizol method
RNA isolation by Trizol method is showing after adding chloroform and centrifuging, the mixture separates into 3 phases with the upper clear aqueous phase containing the RNA, inter phase containing cell debris and lower is organic phase having protein, lipids, etc. as shown above image.