Acid+Guanidinium-Thiocyanate-Phenol-Chloroform+Extraction+of+Total+RNA

Acid Guanidinium-Thiocyanate-Phenol-Chloroform Extraction Introduction The acid guanidinium-thiocyanate-phenol-chloroform extraction of RNA is used to extract and purify RNA samples from ribosomes and protect the RNA from RNases, compounds that would degrade RNA. The test is best used for the extraction of whole RNA from the cells. The protocol in the original experiment used mammal tissue, but the extraction has been used for over twenty years by different researchers working with plant, animal, and cultured cell tissues. The technique is available commercially under a variety of names, but it can be prepared in the laboratory. If the solutions are made in the lab, it should be noted that some of the compounds used are toxic and can be dangerous if mishandled.

List of Reagents  Reagent Preparation dissolve 250 g 4 M guanidinium thiocyanate in 293 mL water at 65 C. To this mixture, add 17.6 mL 0.75 M sodium citrate, pH 7. Then add 26.4 mL of 10% (wt/vol) Sarkosyl. This is the stock solution. To 50 mL stock solution, add 0.36 mL 98% 2-Mercaptoethanol. This is Solution D, the denaturing solution. This solution will denature the proteins and ribosomes and free the DNA and RNA.
 * Guanidinium thiocyanate
 * Sodium citrate
 * N-laurylsylsarcosine (Sarkosyl)
 * 2-Mercaptoethanol
 * anhydrous Sodium acetate
 * Glacial acetic acid
 * nucleic acid-grade Phenol
 * Chloroform
 * Isoamyl alcohol
 * Isopropanol
 * Ethanol
 * Diethylpyrocarbonate (DEPC)
 * Sodium dodecyl sulfate (SDS)

Denaturing and Extraction The tissue of interest should now be minced and homogenized in 1 mL of solution D, either with a glass-Teflon homogenizer or a power homogenizer. The homogenate should be transfered to a 4 mL polypropylene tube and inverted. Then add 0.1 mL 2 M sodium acetate, pH 4. The tube should be inverted again to mix. Then add 1 mL of water-saturated phenol (this will be used to create an aqueous an organic layer to separate RNA from DNA). Invert the tube again. Add 0.2 mL chloroform-isoamyl alcohol mixture (49:1). Shake the tube vigorously for 10 seconds. Cool this mixture on ice for 15 minutes. After this, centrifuge at 10,000 gravities for 20 minutes at 4 degrees C. The liquid will be separated into an aqueous and organic phase. The RNA should be in the aqueous phase and the DNA should be in the organic phase with the phenol. Add the upper aqueous phase to a clean tube using a pipette. Add 1 mL isopropanol to precipitate the RNA. Incubate the samples for 1 hour at -20 degrees C. At this point, the RNA can be stored and the experiment can be performed later, or the experiment can continue after the hour is up. After, the tube is centrifuged again for 20 minutes at 10,000 gravities at 4 degrees C. The RNA should be precipitated at the bottom of the tube in the form of a pellet. The rest of the liquid (supernatant) can be discarded. Dissolve the RNA pellet in 0.3 mL solution of solution D. Transfer the solution to a 1.5 mL microcentrifuge tube. Add 0.3 mL isopropanol. Incubate at -20 degrees C for 30 minutes. At this point the experiment can be halted and again and the samples stored. After, centrifuge at 10,000 gravities for 10 minutes at 4 degrees C. Resuspend the new RNA pellet with 0.5-1 mL 75% ethanol and vortex for a few seconds. Incubate at room temperature for 10-15 minutes to dissolve traces of guanidinium. Centrifuge for 5 minutes at 10,000 gravities at 4 degrees C and discard the supernatant. Air dry the pellet for 5-10 minutes. Do not let the pellet dry completely. Dissolve the pellet in 100-200 microliters of DEPC-treated water or 0.5 SDS. Incubate at 60 degrees C for 10-15 minutes. The sample is ready to be quantified.

Quantification The quantification can be obtained using spectrophotometric readings. This will establish the purity of the sample. The wavelengths should be measured at 260 nm and 280 nm. At 260 nm, an optical density of 1.0 corresponds to 40 micrograms/mL of RNA. A ratio of 1.8-2.0 of absorbance at 260/absorbance at 280 will indicate pure RNA. A lower ration indicates contamination.

Cited Literature The procedure described above is from the article that first describes this method and its reissue, which updates some of the procedures. If anything has been left out, or if something is unclear, the original papers can explain in more detail. All the credit goes to the researchers who developed this method.


 * 1) Chomcynski P., Sacchi N. Single-step Method of RNA Isolation by Acid Guanidinium-Thiocyanate-Phenol-Chloroform Extraction. Analytical Biochemistry 1987 Apr; 162(1) 156-159.
 * 2) Chomcynski P., Sacchi N. The Single-step Method of RNA Isolation by Acid Guanidinium-Thiocyanate-Phenol-Chloroform Extraction: Twenty-Something Years on (Ribonucleic Acid). Nature Protocols 1.2 (July 2006): p581(5).