RNA Extraction with CsCl

Dominik Haudenschild, Genzyme Tissue Repair, August 11, 1997

Use for difficult tissues, such as Cartilage.

Homogenization Buffer

4.0M Guanidium thiocyanate (mw 118.16)>
0.1M Tris-HCl (pH 7.5)
Filter sterilize, store up to 1 yr RT
Just before use, add ß-mercaptoethanol to 1% (v/v)

Cesium Chloride/EDTA

5.7M Cesium Chloride
10mM EDTA
Add 0.1% DEPC, allow to stand ³20min
Autoclave 20 minutes

Homogenize Tissue

Freeze cartilage in -80°C freezer or nitrogen, weigh

Add dry ice
Grind in coffee grinder or blender until powdery consistency
Add 5 volumes homogenization buffer to frozen tissue, (or 5ml/T225)
Wait until dry ice boils away
Use homogenizer to grind tissue
Add Sodium Lauryl Sarcosinate to 0.5%, mix well
Spin to pellet insoluble clumps (5000g, 10 min)
Re-extract clumps with 5 additional volumes of homogenization buffer

Centrifuge

Pull homogenized tissue into syringe with 23 gauge needle
Layer up to 2 volumes of homogenized tissue onto CsCl cushion in tube
Mark position of top of CsCl cushion with waterproof pen

Rotor

CsCl Vol.

Tissue Vol.

Time (hrs)

Speed (rpm)

SW-60

1.2

3.1

12

40,000

SW-40

3.5

9.7

24

32,000

SW-28

12

26.5

26

23,500
Spin as indicated below, at 20°C
Mark tubes 0.5cm from bottom
Gently, with pasteur pipet, remove fluid above cushion layer (DNA = white band)
With fresh pipet, remove remaining fluid down to lowest mark on tube
With red-hot razor blade, cut tube just above liquid level
Gently aspirate out remaining liquid
Invert tube onto clean pad of Kimwipes (make sure pellet remains in tube)
Fill bottom of tube with 70% ethanol in DEPC water
Invert tube, drain ethanol into Kimwipes
Air-dry pellet
Dissolve in TE with 0.1% SDS (300µl per 10ml homogenized tissue)
Transfer to microcentrifuge tube,
Precipitate with Sodium acetate (pH 5.2) and Ethanol
Wash pellet with 70% Ethanol in DEPC water
Store in small amount of TE or DEPC Water