RNA Extraction from Blood

1. Introduction

Based on the method of Chomczynski and Sacchi (1), this is an extremely reliable

method without the requirement for centrifugation over CsCl gradients. As with any

RNA protocol, extreme care should be taken to exclude RNAse contamination, the

greatest source of which will be the sample itself. All disposables and reagents should

be RNAse free.

2. Materials

1. Microfuge tubes (1.5 mL).

2. Ice bucket.

3. Microfuge.

4. Red cell lysis buffer: 1.6 M sucrose, 5% Triton X-100, 25 mM MgCl2, 60 mM Tris-HCl,

pH 7.5; stored at 2–8°C and used cold.

5. Extraction Buffer: 5.25 M guanidinium thiocyanate, 50 mM Tris-Cl, pH. 6.4, 20 mM

EDTA, 1% Triton X-100, 0.1 M â-mercaptoethanol (add immediately prior to use).

6. 2 M sodium acetate, pH 4.0.

7. Phenol (saturated with 1 M Tris-HCl: 0.1 M EDTA, pH 8.0).

8. Chloroform:Iso-amyl alcohol (24��1).

9. Isopropyl alcohol.

10. 70% Ethanol.

11. RNAse-free distilled water.

3. Method

1. In a microfuge tube, mix 100 ìL anticoagulated blood with 1 mL of red cell lysis buffer

(see Notes 1–3).

2. Leave at room temperature with occasional shaking until the red cells have lysed and the

solution translucent (usually within 5 min).

3. Microfuge for 30 s at 13,000g to pellet the white blood cells. Remove and discard

supernatant.

4. Add 200 ìL of extraction buffer and resuspend cell pellet by drawing through narrow

gauge needle several times.

5. Add 20 ìL of 2 M sodium acetate and mix gently by inversion.

6. Add 220 ìL of phenol and mix gently by inversion.

7. Add 60 ìL of chloroform/isoamyl alcohol (24��1) and vortex vigorously.

8. Place on ice for 15 min.

9. Microfuge at 12,000g for 5 min and transfer the upper phase to new microfuge tube.

10. Add 200 ìL of ice-cold isopropanol mix and store at –20°C for 30 min.

11. Microfuge at 12,000g for 15 min and discard supernatant.

12. Resuspend pellet in 200 ìL of extraction buffer.

13. Repeat steps 3 through 9.

14. Wash pellet with 400 ìL of cold 70% ethanol.

15. Microfuge at 12,000g for 5 min and discard supernatant.

16. Carefully remove last traces of ethanol from tube (folded sterile swab or kimwipe works

well).

17. Resuspend in 100 ìL of distilled water and incubate at 50°C for 15 min to dissolve RNA

(see Note 4).

4. Notes

1. Blood stored at room temperature or 4°C should be mixed thoroughly prior to aliquots

being removed.

2. Frozen blood samples should be allowed to thaw completely and mixed thoroughly before

aliquots being removed. Although freezing lyses red blood cells, the red cell lysis step

should still be performed to efficiently remove hemoglobin from the sample. Repeated

freeze/thaw cycles should be avoided.

3. Buffy coat contains two to four times the amount of white blood cells per volume compared

to fresh blood. Therefore, it is advisable to use only 50 ìL of buffy coat diluted with 50 ìL

of phosphate-buffered saline as starting material for this protocol.

4. Repeat pipetting through a narrow gauge tip can help this process.