Because some tissues (e.g., pancreas and uterus) contain high concentrations of endogenous RNAases, more care must be taken in purifying tissue-derived RNA.

Operation method

RNA extraction from tissues by CsCl method

Materials and Instruments

Liquid nitrogen Tissue guanidine solution Sodium dodecyl sarcosinate CsCl Tissue resuspension solution Sodium acetate Phenol Chloroform Isoamyl alcohol Isoamyl alcohol Anhydrous ethanol
Tissue masher Centrifuge

Move

I. Materials and equipment

1）Liquid nitrogen

2) Tissue guanidine solution: 590.8 g of guanidine isothiocyanate was dissolved in 400ul of DEPC-treated water, 25 mmol/LTris-Cl (pH 7.5) was added.20 ml of 0.5 mol/LEDTA (pH 8.0) was stirred overnight, water was added to 950 ml, filtered, and finally 50 ml of, β-mercaptoethanol was added.

3) 20% (m/V) sodium dodecyl sarcosinate

4) 5.7 mol/LCsCl

5) Tissue resuspension:5 mmo/LlEDTA, 0.5% (V/V) sodium dodecyl sarcosinate, 5% β-mercaptoethanol

6) 3 mol/L sodium acetate pH5.2

7) Phenol: chloroform: isoamyl alcohol (25:24:1), chloroform: isoamyl alcohol (24:1)

8) Tissue masher

9) Centrifuge

10) Anhydrous ethanol.


II Methods of operation

1) Quickly remove the tissue from the animal and cut it into small pieces of less than 2 g. Quickly freeze in liquid nitrogen. Quickly freeze in liquid nitrogen, add 20 ml of tissue guanidine solution (without sodium dodecyl sarcosinate) per 2 g of tissue and immediately grind in a tissue masher 2-3 times for 10s each time. Quick freezing is critical because RNA degradation occurs when the tissue is placed in guanidine solution and waiting to be mashed.

2) Centrifuge the tissue at 12OOOg for 10 mim at 12°C. Add 0.1 times the volume of 20% sodium dodecyl sarcosinate to the supernatant and heat at 65°C for 2 min.

3) Add O.lgCsCl per ml of liquid and allow to dissolve. Add the sample to a 9 ml cushion of 5.7 mol/LCsCl in a centrifuge tube that has been autoclaved and siliconized. Centrifuge at 113,000 g at 22°C overnight.

4) Carefully remove the supernatant. Invert the tube to drain the remaining liquid. Cut off the bottom of the centrifuge tube (containing the RNA precipitate) and place it in a 50 ml plastic tube with 3 ml of Tissue Resuspension Solution at 4°C to dissolve the precipitate overnight or longer.

5) Extract the solution sequentially with phenol: chloroform: isoamyl alcohol (25:24:1) and chloroform: isoamyl alcohol (24:1).

6) Add 0.1x volume of 3moI/LPH5.2 in sodium acetate buffer and 2.5x volume of anhydrous ethanol, precipitate, redissolve RNA in water, quantify and store.

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