Isolation of RNA by Trizol method :-

*Principle:-

RNA extraction is the purification of RNA from biological samples. ...
Acid guanidinium thiocyanate-phenol-chloroform extraction is a liquid–liquid extraction technique in biochemistry. It is widely used in molecular biology for isolating RNA.
 The most common of these is guanidinium thiocyanate-phenol-chloroform extraction,
followed by centrifugation.
Under acidic conditions, total RNA remains in the upper aqueous phase, while most of DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for several applications.
Acid guanidinium thiocyanate-phenol-chloroform extraction is a liquid–liquid extraction technique in biochemistry. It is widely used in molecular biology for isolating RNA.

*Protocol :-


1| Add 1 ml of Trizol to your suspension of mononuclear cell sample and pipette extensively for the liquid to become transparent
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2| Add 200 µl of chloroform. Shake well.
                    ⬇️
                    
3| Centrifuge at max speed for 20 minutes at +4.
                   ⬇️

4| Transfer the tubes on ice. While keeping the tube cool, transfer the upper phase into a new tube.
                   ⬇️

5| Add 1 µl of co-precipitant (optional) and 500 µl isopropanol.
                   ⬇️
                   
6| Centrifuge at max speed for 20 minutes at 4°c.
                  ⬇️
                  
7| Discard the supernatant.
                  ⬇️

8| Add 200 µl 80% EtOH.
                  ⬇️
                  
9| Centrifuge at max speed for 2 minutes at 4°c.
                   ⬇️
                   
10| Repeat steps 7-9 (optional).
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11| Discard the supernatant.

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12) Place the tube opened at 50 degrees. As soon as EtOH evaporates take the tube off the thermostat.
                    ⬇️

13) Dilute RNA in steril RNAse free water.
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14| Check the amount and quality of RNA using gel-electrophoresis.

*NOTE :-
_ Improper ratio Trizol reagent: sample may lead to insufficient cell lysis and affect the RNA yield. Use correct amount of lysis reagent. For example, use 1 ml of Trizol per up to 10^7 cells.
_ Do not overdry RNA - it leads to a decrease in recovery.

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