Trizol

Protocol 1

1. Harvest cells, spin down cells gently, add an approximately equal pellet volume of 1X PBS and suspend cells
2. If you freeze cell pellet on dry ice, do not add 1XPBS, add Trizol directly to frozen pellet, then mix vigerously.
3. Add 1ml TRI Reagent (for between 10^5 and 3 x 10^7 cells) and suspend cells immediately.
4. Incubate at room temperature for 5-15 minutes.
5. Add 200ul CHCl3, vortex vigorously for 10-20 seconds.
6. Incubate at room temperature for 5 minutes.
7. Spin in at maximum speed for 15 minutes.
8. Transfer aqueous phase into a new tube
9. Add 0.5ml isopropanol, mix, and keep at room temperature for 10 minutes.
10. Spin at max speed for 20 minutes.
11. Take off (or aspirate) the aqueous/isopropanol solution.
12. Wash with 800 ul 70% EtOH (stored at –20oC)[i.e. add 70% EtOH, vortex, spin 5 minutes (high speed in microfuge) and take off EtOH]
13. Dry in speedy vac and suspend RNA in 50-250 ul molecular biology grade H2O.

Homemade Trizol Reagent (Acid Guanidinium Thiocyanate-Phenol-Chloroform) solution

- 4M Guanidinium thiocyanate: add 293 ml H2O to 250 g guanidinium thiocyanate (Fluka)
- 0.75 M Sodium citrate pH 7.0
- 2 M Sodium Acetate pH 4.0
- 10% Sarcosyl (1g/10ml H2O and filter)
- Phenol (nucleic acid grade, Sigma) (warm phenol bottle at 65 oC, add 0.1% w/v hydroxyquinoline and saturate with H2O)

21 ml of TRIzol reagent
- Mix 10 ml 4M guanidinium thiocyanate, 352ul 0.75M sodium citrate (pH 7.0) and 528ul 10% sarcosyl
- Add 76 µl 14.3 M ß-mercaptoethanol (Sigma), total final volume is 10.9 ml.
- Place 10 ml of this mixture in a new tube, add 1 ml 2M sodium acetate (pH 4.0) and 10 ml water saturated phenol.

105 ml of TRIzol reagent
- Mix 50 ml 4M guanidinium thiocyanate, 1.76 ml 0.75M sodium citrate (pH 7.0) and 2.64ml 10% sarcosyl
- Add 381 µl 14.3 M ß-mercaptoethanol (Sigma), total final volume is 54.5 ml.
- Place 50 ml of this mixture in a new tube, add 5 ml 2M sodium acetate (pH 4.0) and 50 ml water saturated phenol.

Store TRIzol reagent at 4 oC. It is stable for at least 2 months.

1L Trizol

Reagents Volume/mass Final concentration
Acid Phenol 380mL 38%
Guanidine thiocyanate 94.53g 0.8M
Ammonium thiocyanate 30.45g 0.4M
Sodium acetate, pH 5.0 33.4mL of 3M stock 0.1M
Glycerol 50mL 5%
RNase-free water Adjust final volume to 1L

Other reagents:
DEPC-Water Adjust the final volume to 1 L
0.8 M sodium citrate / 1.2 M NaCl
Isopropanol (2-Propanol)
Chloroform
DEPC-Water
75% ethanol prepared with DEPC-Water

Protocol 2

Material

TRIzol Reagent.

0.8 M sodium citrate / 1.2 M NaCl

Isopropanol (2-Propanol)

Chloroform

DEPC-Water

75% ethanol prepared with DEPC-Water

RNase Inhibitor (e.g., aseERASE, BIO 101 Cat. 2601-104)
50 mL sterile plastic screw-cap centrifuge tubes

1. Grind 1g tissue in liquid nitrogen in a mortar and pestle.

2. Transfer powdered tissue to a 50 mL sterile plastic screw-cap centrifuge tube containing 15 ml TRIzol reagent. Incubate samples at room temperature or at 60°C for 5 min.

3. Homogenize tissue with homogenizer for 15 seconds. Repeat once.

4. Centrifuge samples at 12,000 x g at 4°C for 10 min.

5. Transfer supernatant into new sterile 50 mL sterile plastic screw-cap centrifuge tube. Discard pellet.

6. Add 3 mL chloroform to each tube in hood. Shake tubes vigorously with vortex for 15 sec.

7. Let tubes sit at room temp 2-3 min. Centrifuge tubes at 10,000 x g at 4°C for 15 min.

8. Carefully pipet aqueous phase into a clean screw-cap centrifuge tube; discard interphase and lower phase into waste.

9. Precipitate RNA by adding isopropanol and 0.8 M sodium citrate/1.2 M NaCl, half volume of the aqueous phase each. Cover tube and mix by gentle inversion. Let sit at room temperature for 10 min.

10. Centrifuge tubes at 10,000 x g at 4°C for 10 min. Discard supernatant.

11. Wash pellet with 20 ml of 75% ethanol. Vortex briefly.

12. Centrifuge at 10,000 x g at 4°C for 10 min. Discard supernatant; briefly dry pellet on kimwipe.

13. Add 100-250 µL DEPC-Water, to pellet. Resuspend RNA by pipetting up and down a few times.

14. Add 1 µL RNase inhibitor aseERASE to a 250 µL RNA sample. If having problems resuspending the RNA pellet, we suggest incubation at 55 - 60°C for 10 min.

15. Transfer sample to microcentrifuge tube at room temperature.

16. Spin samples at high speed in microcentrifuge tube for 5 min at room temperature (to pellet the material that would not resuspend).

17. Transfer RNA solution (supernatant) to a new tube. Determine RNA concentration and quality by spectrophotometry.

Note: For optimal spectrophotometric measurements, RNA aliquots should be diluted with water or buffer with a basic pH. Water with pH< 7.5 falsely decreases the 260/280 ratio.

Reagents	Volume/mass	Final concentration
Acid Phenol	380mL	38%
Guanidine thiocyanate	94.53g, or 118.16 g?	0.8M
Ammonium thiocyanate	30.45g, or 76.12 g?	0.4M
Sodium acetate, pH 5.0	33.4mL of 3M stock	0.1M
Glycerol	50mL	5%
DEPC treated RNase-free water	Adjust final volume to 1L

Protocol 3

Solution D (based on Chomczynski and Sacchi1987/2006) is:

• 4 M guanidinium thiocyanate
• 25 mM sodium citrate
• pH 7.0
• 0.5% (wt/vol) N-laurosylsarcosine (Sarkosyl)
• 0.1 M 2-mercaptoethanol

Prepare stock with:

• dissolving 250 g guanidinium thiocyanate in 293 ml water at 65 °C
• add 17.6 ml of 0.75 M sodium citrate, pH 7.0
• 26.4 ml of 10% (wt/vol) N-laurosylsarcosine

(stored <3 months at room temperature)

Working solution from stock:

• add 0.36 ml of 98% 2-mercaptoethanol to 50 ml of stock solution

(store <1 month at RT)

Using Solution D

Assuming you start with 10 million cells or 100 mg of tissue (keep cool when possible):

• add 1 ml solution D (don't linger on this step)
• transfer to tubes
• add 0.1 ml of 2 M sodium acetate, pH 4.0, and invert tube to mix
• add 1 ml water-saturated phenol (never buffered phenol) and invert tube
• add 0.2 ml of chloroform/isoamyl alcohol (49:1) and shake vigorously for 10 sec
• centrifuge 20min 10000G 4ºC
• transfer top aqueous phase into new tube
• precipitate