Isolation of rna from yeast procedure steps

The isolation of RNA from yeast involves breaking open the tough yeast cell wall. It also involves ensuring the RNA is protected from degradation by RNases.

Here is the step-by-step procedure of rna isolation from yeast.

Materials Needed for Isolation of rna

1. Yeast cells (grown to the desired OD in culture).
2. RNA extraction reagent (e.g., TRIzol or similar).
3. Phenol:Chloroform:Isoamyl Alcohol (25:24:1 mixture).
4. Isopropanol (for RNA precipitation).
5. 70% Ethanol (for washing RNA pellet).
6. RNase-free water (to dissolve RNA).
7. Glass beads (0.5 mm diameter, for cell lysis).
8. Bead beater or vortex with bead-beating adapter.
9. Microcentrifuge.
10. RNase-free tubes.

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Isolation of RNA from yeast procedure

Step 1: Yeast Cell Harvesting

1. Grow yeast cells to the desired phase (e.g., log phase) and measure OD600.
2. Harvest cells by centrifugation at 3,000 x g for 5 minutes.
3. Wash the pellet once with RNase-free water to remove residual media.
4. Resuspend the pellet in a small volume of RNase-free water or RNA extraction reagent.

Step 2: Cell Lysis

1. Add an appropriate volume of RNA extraction reagent (e.g., TRIzol) to the resuspended cells.
2. Add an equal volume of glass beads (0.5 mm) to the tube.
3. Vortex vigorously using a bead beater or vortex for 5-10 minutes. Alternate between vortexing and resting on ice. This prevents overheating.
   • Ensure the cells are completely lysed (the solution will become viscous if RNA is released).

Step 3: Phase Separation

1. Centrifuge the lysate at 12,000 x g for 10 minutes at 4°C to pellet debris.
2. Transfer the supernatant to a new RNase-free tube.
3. Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1).
4. Mix thoroughly by inverting the tube, then centrifuge at 12,000 x g for 10 minutes at 4°C.
   • The RNA will remain in the aqueous (upper) phase.

Step 4: RNA Precipitation

1. Carefully transfer the aqueous phase to a new RNase-free tube.
2. Add 0.5 volumes of isopropanol to the aqueous phase.
3. Mix gently and incubate at -20°C for 30 minutes to precipitate the RNA.
4. Centrifuge at 12,000 x g for 10 minutes at 4°C to pellet the RNA.

Step 5: Washing the RNA Pellet

1. Wash the RNA pellet with 70% ethanol (made with RNase-free water).
2. Centrifuge at 7,500 x g for 5 minutes to remove residual ethanol.
3. Carefully discard the ethanol and air-dry the pellet (do not over-dry).

Step 6: RNA Resuspension

1. Resuspend the RNA pellet in a small volume (20-50 µL) of RNase-free water.
2. Store at -80°C or proceed to downstream applications.

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How to check the quality and purity of RNA

1. Measure RNA concentration and purity using a spectrophotometer (e.g., NanoDrop).
   • Check the A260/A280 ratio (should be ~2.0 for pure RNA).
   • Check the A260/A230 ratio (should be >2.0).
2. Verify RNA integrity on an agarose gel or using a bioanalyzer.

This procedure ensures high-quality RNA suitable for applications like RT-PCR, RNA-seq, or Northern blotting.