Cat #: Bio-Hi                           

Size: 100 ml

Description:  TriSolution Reagent is a mixture of phenol, guanidium thiocynate, buffers and stabilizers developed for the simultaneously isolation of total RNA, DNA and Proteins from animal and plant tissue, cells and bacteria culture. The entire procedure for total RNA isolation is an improvement single-step method developed by Chomczynski and Sacchi.  After

homogenization of sample and chloroform extraction, three phases are formed (aqueous phase, interphase and organic phase).  RNA can be precipitated by isopropanol from aqueous phase, DNA can be recovered by ethanol precipitation from interphase, and proteins are precipitated with isopropanol from organic phase.

General Procedure for RNA Isolation :

Materials to be supplied by the user:

Chloroform, chilled

Isopropanol, chilled

75% Ethanol ( in DEPC-treated water or RNase- free water )

1. HOMOGENIZATION

  a.Tissues

     Homogenize tissue samples in 1 ml of Hi.Extraction per 50-100 mg of tissue.

  b.Cells Grown in Monolayer

     The amount of Hi.Extraction added is based on the area of the culture dish (1 ml per 10 cm²

      and not on the number of cells present.

  c.Cells Grown in Suspension

     Pellet cells by centrifugation. Lyse cells in Hi.Extraction by repetitive pipetting. Use 1 ml of

     the reagent per 5-10 × 10⁶ of animal, plant or yeast cells, or per 1 × 10⁷ bacterial cells.

     Washing cells before addition of Hi.Extraction should be avoided as this increases the

     possibility of mRNA degradation. Disruption of some yeast and bacterial cells may require the

     use of a homogenizer.

2.  Incubate the samples for 5 minutes at RT to allow the complete dissociation of nucleoprotein

     complexes.

3.  Add 0.2 ml of chloroform per 1 ml of Hi.Extraction. Cap sample tubes securely.

4.  Shake tubes vigorously by hand for 15 seconds and incubate them at RT for 3 minutes.

5.  Centrifuge the samples at 12,000 × g for 15 minutes at 4°C

6.  Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an

     interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous

     phase. The volume of the aqueous phase is about 60% of the volume of Hi.Extraction used for

     homogenization.

7.  Transfer the aqueous phase to a fresh tube, Use 0.5 ml of isopropyl alcohol per 1 ml of

     Hi.Extraction used for the initial homogenization.

8.  Incubate samples at RT for 10 minutes.

9.  Centrifuge at 12,000 × g for 10 minutes at 4°C.

10. Remove the supernatant, adding at least 1 ml of 75% ethanol per 1 ml of Hi.Extraction used

      for the initial homogenization. Mix the sample by vortexing.

11. Centrifuge at 7,500 × g for 5 minutes at 4°C.

12. Briefly dry the RNA pellet (air-dry or vacuum-dry for 10 minutes).

13. Partially dissolved in RNase-free water or 0.5% SDS solution and stored at -70°C.