Principal
RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. Several methods are used in molecular biology to isolate RNA from samples, the most common of these is guanidinium thiocyanate-phenol-chloroform extraction. The filter paper based lysis and elution method features high throughput capacity.
RNA extraction in liquid nitrogen, commonly using a mortar and pestle is also useful in preventing ribonuclease activity.
TRIzol is a widely used chemical solution used in the extraction of DNA, RNA,and proteins from cells. The solution was initially used and published by Piotr Chomczyński and Nicoletta Sacchi in 1987. TRIzol is the brand name of guanidinium thiocyanate The correct name of the method is guanidinium thiocyanate-phenol-chloroform extraction. The use of TRIzol can result in DNA yields comparable to other extraction methods, and it leads to greater than 50% bigger RNA yield.
TRIzol is light-sensitive and is often stored in a dark-colored, glass container covered in foil. It is stored at room temperature.
When used, it resembles cough syrup, bright pink. The smell of the phenol is extremely strong. TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components.
Procedure
I. Homogenization
1. Prepare TRIzol reagent in a 50 ml Screw Cap tube at room temperature RT before taking the sample out as described in the table. For 50-100 mg sample 1 ml trizol reagent.
2. Cut the sample off into small pieces approximately 50-100 mg
3. Immediately place into TRIzol reagent in 50 ml Screw Cap tube, and homogenize using a Tissue Homogenizer for 30- 60 seconds.
4. Incubate at RT Room Temperature for 5-10 minutes after homogenization.
II. Phase separation.
5. Transfer the homogenate to 2 ml Centrifuge tube and add 1 ml of trizol reagent. Incubate for 10 min at 37 degree Celsius and add 1 ml chloroform to it. Shake tube vigorously for 15- 30 seconds by hand
6. Centrifuge at 10,000 rpm for 20 minutes at 4ºC.
7. Carefully remove the upper aqueous phase which contains the total RNA, and place this in a new 2 ml centrifuge tube.
8. Preserve bottom layer at 4ºC for subsequent isolation of DNA and proteins.
III. RNA precipitation and wash.
9. . Add equal amount of Chilled Lithium Chloride as per the aqueous layer obtained incubate RT 20 min.
10. Centrifuge 10,000 rpm for 8 min at 4ºC.
11. Wash the pellet with at least 1 ml of 70 Percent Ethanol
12. Centrifuge 10,000 g for 5 min at 4ºC.
13.Wash one more time with 70% Ethanol.
14. Dry the pellet at RT do not dry completely and re-suspend the RNA pellet in 200 –300 µl DEPC –TE.
15. Sample is ready for any analysis or Agarose Gel electrophoresis.
