This protocol was published by Godfrey et al. as an improvement on Fisher's protocol. Previously, protocols for purifying RNA from fixed tissues have been published, and commercial kits for purifying RNA from paraffin-embedded tissues have been invented by Ambion and Roche. This experiment was derived from PCR Laboratory Guide (Second Edition) by Kang Seed and Lijia Qu.

Operation method

Preparation of RNA from paraffin-embedded fixed tissues

Materials and Instruments

Paraffin-embedded tissue samples
Formamide Digestive buffer Ethanol Hepatose Isopropyl alcohol Phenol Chloroform Protease K Trizol Xylene
Microcentrifuge tubes Thermostat

Move

I. Materials

1. Buffers, solutions and reagents

Deionized formamide

Diethyl ethyl pyrocarbonate (DEPC) Treated water

Digestion buffer (lmol/L guanidine isothiocyanate, 25 mmol/L β-mercaptoethanol, 0.5% N-lauroyl sarcosine, 20 mmol/L Tris-HCl, pH 7.5)

Ethanol, 100% and 70

Hepatose

Isopropyl alcohol

Phenol (pH 4.3): Chloroform (70%:30%)

Proteinase K (60 mg/ml, 20U/mg)

Trizol (Invitrogen)

Xylene

2. Specialized equipment

Microcentrifuge tube, 2 ml, without RNAase

Thermostat, pre-conditioned to 37°C

Water bath or thermostat, pre-conditioned to 55°C

3. Cells and tissues

Paraffin-embedded tissue samples, cut into 5-50 5um thick sections

II. Methods

1. Place the tissue block in a 2.0 ml microcentrifuge tube and add 1.8 ml xylene to the sample. incubate at 37°C for 20 min.

2. Slightly centrifuge the sample, discard the supernatant and add xylene, repeat the incubation and centrifugation steps.

3. Wash the tissue with 0.5 ml ethanol. Discard the ethanol and air dry.

4. Add 80ul of Proteinase K (60 mg/ml, 20U/mg) and 72ul of Digestion Buffer. incubate overnight at 55°C with shaking.

5. Add 80ul of Proteinase K again and incubate overnight at 55°C with shaking. The next day, 80ul of Proteinase K was added and incubated again at 55°C overnight with shaking.

6. add equal volumes of phenol: chloroform, mix, and centrifuge in a microcentrifuge at maximum speed for 5 min. transfer the liquid phase to a new tube for RNAase.

7. Add equal volumes of isopropanol and 2ug of heparanose and incubate at -20°C for 30 min.

8. centrifuge in a microcentrifuge at maximum speed for 30 min. remove the supernatant and wash the sediment with 70% ethanol.

9. centrifuge in a microcentrifuge at maximum speed for 30 min and discard the supernatant. Air dry the supernatant and redissolve it in 20ul of DEPC treated water. Follow the Trizol protocol (see Protocol 3 in this chapter) with one modification: after the first Trizol treatment, transfer the liquid phase to a new RNAase-free tube and treat the sample a second time with 500ul of Trizol.

10. Add 500ul of isopropanol to the second Trizol-treated liquid phase to precipitate the RNA.

11. Redissolve the RNA precipitate in 20ul of deionized formamide. Store RNA at -20°C.

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