RNA EXTRACTION TECHNIQUE by using TRIzol

       TOTAL RNA EXTRACTION VIA TRIzol TECHNIQUE

           TRIzol reagent is designed to isolate high quality total RNA from tissue samples of human ,     animal ,plant, yeast , or bacteria origin ,with in one hour .

   composition of TRIzol

                                      Phenol  +  guanidine isothiocyanate

   Material Required:

                            Centrifuge Machine, Polypropylene microcentrifuge tubes, Chloroform reagent, 

 Isopropanol, Ethanol 75%, RNase free distilled water, TRIzol reagent, micropippets ,Micro Tips, eppendorf tubes

   Procedure:

when we are taking sample of  a tissue structure we will use this procedure.

1.  Add 1 mL of TRIzol Reagent per 50  to 100 mg of tissue sample and homogenize it using a homogenizer.

2.Add 0.2 mL of chloroform per 1mL of TRIzol Reagent used for lysis ,then securely cap the tube.

3.Incubate for 2_3 minutes.

4.Centrifuge the sample for 15 minutes at 12000 rpm at 4 degree celsius.

   The mixture separates into a lower red phenol-chloroform, and interphase ,and a colorless upper aqueous phase.

5.Transfer the aqueous phase containing the RNA to a new tube.

6.Add 0.5 mL of isopropanol to the aqueous phase .

7.Incubate for 10 minutes.

8.Centrifuge the sample for 15 minutes at 12000 rpm at 4 degree celsius.

Total RNA precipitate forms a white gel like pellet at the bottom of the tube.

9.Discard the supernatant with a micropipett. 

10.Resuspend the pellet in 1 mL of 75% ethanol .

11.Vortex the sample briefly then centrifuge for 5 minutes at 7500 rpm at 4 degree celsius.

12..Discard the supernatant with a micropipett. 

13.Resuspend the pellet in 20-50 micro L of RNase free  water or 0.1mM EDTA .

            NOTE

                           Isolated RNA can be used in R-T PCR ,Northern blot analysis,Dot Blot Hybridization poly (A)+selection , in vitro translation,RNase protection assay and molecular cloning.