The production of phenol-based reagents for RNA isolation is based on Chomczynski protocols. These protocols are most useful when obtaining RNA from RNAase-rich tissues such as pancreas. Presented here is a summary of the protocols sold with Invitrogen's Trizol reagents, modified with Invitrogen's permission. This experiment was derived from PCR Laboratory Guide (Second Edition) by Kang Seed and Lijia Qu.

Operation method

RNA purification experiments with Trizol

Materials and Instruments

Finely ground tissue
Trizol Chloroform Isopropyl alcohol Ethanol DEPC treated water

Move

I. Materials
1. Buffers, solutions and reagents
Trizol (Invitrogen)
Chloroform
Isopropyl alcohol
Ethanol, 75%, prepared with diethyl pyrocarbonate (DEPC) treated water
DEPC-treated water
2. Cells and tissues
50-100 mg finely ground tissue
II. Methods
1. Add lml Trizol to a microtube containing 50-100 mg of finely ground tissue. Allow to incubate for 5 min at room temperature.
It is important that the tissue is finely ground to help Trizol cleave it.
2. Add 200ul of chloroform and invert the tube several times to mix.
Shaking is not recommended as it can lead to shearing of genomic DNA and excessive DNA contamination in the RNA preparation.
3. incubate at room temperature for 3 min and centrifuge at 1500 g for 15 min.
4. Transfer the liquid phase to a new tube. Add 500ul of isopropyl yeast. Incubate at room temperature for l0 min.
5. Centrifuge at 1500 g for l0miri. RNA will be visible at the bottom of the tube.
6. Dispose of the supernatant, taking care not to disturb the sediment.
7. Add 1 ml of 75% ethanol and resuspend the RNA. Resuspend the RNA. Centrifuge at 1500 g at 4°C for 5 min.
8. Remove ethanol and air dry the RNA.
9. Dissolve RNA in DEPC-treated water.
When using phenol-based reagents, a second round of phenol-chloroform extraction of the resulting RNA may be necessary to obtain sufficient purity.
When using phenol-based reagents, a second round of phenol-chloroform extraction of the resulting RNA may be necessary to obtain sufficient purity.

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