Acid Guanidinium Thiocyanate RNA Isolation Phenol Chloroform Extraction

Acid Guanidinium Thiocyanate RNA Isolation Phenol Chloroform Extraction

RNA Isolation Method:

Step By Step RNA Isolation Protocol

1. For mammalian tissue culture cells use 1 ml Denaturing Solution per 1 X 107 cells. For Drosophila cells us 1 ml Denaturing Solution per 1 X 108 cells. For embryos or tissue, use 1 ml Denaturing Solution per 100 mg tissue (see Tip #2).
2.  Homogenize the tissue or cell culture cells using a Teflon-glass homogenizer or a polytron homogenizer.
3. Transfer the homogenate to a 5 ml polypropylene tube.
4. Add 0.1 ml of 2 M Sodium Acetate and mix thoroughly by inversion.
5.  Add 1 ml of ddH2O Saturated Phenol, mix well by inversion, add 0.2 ml of SEVAG and mix well by inversion.
6.  Incubate the suspension for 15 min at 0° to 4 °C (see Tip #3).
7.  Centrifuge the solution at 10,000 X g at 4°C for 20 min (9,000 rpm using an SS-34 rotor).
8.  Transfer the upper phase (aqueous) to a fresh tube (see Tip #4).
9.  Precipitate the RNA by adding 1 ml (1 volume) of 100% Isopropanol (room temperature).
10.  Place the samples at -20°C for 30 min.
11.  Centrifuge the solution at 10,000 X g at 4°C for 20 min (9,000 rpm using an SS-34 rotor) and discard the supernatant.
12. Dissolve the RNA pellet in 0.3 ml Denaturing Solution and transfer to a 1.5 ml microcentrifuge tube.
13.  To the RNA, re-precipitate the RNA by adding 0.3 ml of 100% Isopropanol and incubating at -20°C for 30 min.
14.  Centrifuge the solution at 10,000 X g at 4°C for 20 min (9,000 rpm using an SS-34 rotor) and discard the supernatant.
15. To the pellet add 1 ml of 75% Ethanol, mix well and incubate for 5 to 10 min at room temperature.
16.  Centrifuge the solution at 10,000 X g at 4°C for 20 min (9,000 rpm using an SS-34 rotor) and discard the supernatant.
17. Resuspend the RNA pellet in 75% Ethanol at room temperature,mix well by inversion and incubate at room temperature for between 5 to 10 min at room temperature.
18.  Centrifuge the solution at 10,000 X g at 4°C for 20 min (9,000 rpm using an SS-34 rotor) and discard the supernatant.
19.  Dry the RNA pellet in a vacuum centrifuge for 5 to 15 min.
20. Dissolve the RNA pellet in 50 to 100 μl ddH2O.
21.  Quantitate the RNA by measuring the absorbance at 260 nm (see protocol on Quantification of RNA) and store in aliquots at -70°C.

RNA Isolation Buffers

ddH2O Saturated Phenol		Combine equal volumes of ddH2O and Phenol (do not use neutralized Phenol) into a brown glass bottle Use the Phenol Mix well and incubate at 4°C overnight
70% (v/v) Ethanol
75% (v/v) Ethanol
SEVAG		24:1 Chloroform:Isoamyl Alcohol
2 M Sodium Acetate		Autoclave pH to 4.0 using Glacial Acetic Acid
10% Sarkosyl		10% (w/v) N-Lauroylsarcosine Sodium Salt
Denaturing Solution		0.1 M 2-Mercaptoethanol* 0.5% (w/v) N-Lauroylsarcosinate Sodium Salt 4 M Guanidinium Thiocyanate (CAUTION! see Tip #1) *Add just before use 25 mM Sodium Citrate, pH 7.0
0.75 M Sodium Citrate

Materials Needed for RNA Isolation Protocol

• Sodium Citrate
• Guanidinium Thiocyanate
• Sarkosyl
• Isoamyl Alcohol
• Ethanol
• Sodium Acetate
• 2-Mercaptoethanol
• Hydrochloric Acid
• Chloroform
• Glacial Acetic Acid
• Phenol

Protocol Tips

Method References