Protocol for Phenol/Chloroform RNA Extraction

Phenol/chloroform extraction is a common technique used to separate and purify DNA, RNA, and protein from a biological specimen, such as a virus preparation. It involves the differential partitioning of DNA/protein and RNA into organic and aqueous phases, respectively. First, a monophasic solution is created by adding acidic phenol and acidic buffer to the nucleic acid-containing sample. The addition of chloroform creates a biphasic system in which DNA and protein partition into the lower organic phase while the upper aqueous phase retains the RNA. Each phase can be subsequently processed to selectively extract the macromolecule of interest.

Graphical Abstract. A graphical representation of the differences between the conventional RNA extraction method and the enhanced RNA extraction method used to avoid RNA contamination. Adapted from “Optimization of phenol-chloroform RNA extraction,” by Toni LS, et al. (2018)., MethodsX, 5, p. 599-608.Click to see expanded image.

RNA isolation protocol using the phenol/chloroform extraction method

Below is a total RNA isolationprotocol using the phenol/chloroform extraction method. For optimal results, special attention should be paid to the removal of RNase contamination. Disposable reagents like microfuge tubes should be sterile and RNase-free. Work surfaces and non-disposable tools and instruments, such as micropipettors and centrifuges, should be treated with an appropriate cleaning agent suitable for eliminating RNases.

1. Sample homogenization and phase separation
1. In a microfuge tube, add 3 volumes of TRIzol LS Reagent or other similar phenol-based solution to 1 volume of liquid sample (e.g., 750 µL of TRIzol + 250 µL of virus preparation).
2. Mix well by pipetting up and down several times.
3. Incubate for 5 min at RT.
4. To the mixture, add 200 µL of chloroform per 750 µL of TRIzol used.
5. Vortex at the maximum setting for 15 sec.
6. Incubate for 3 min at RT.
7. Centrifuge at 12000 × g for 15 min at 4°C.
   At this step, the upper aqueous phase containing total RNA will be separated from the lower organic phase by a yellowish-whitish interphase band.
8. Transfer the upper aqueous phase to a new tube containing fresh chloroform (200 µL of chloroform per 750 µL of TRIzol used).
9. Repeat steps 1e-g.
2. RNA precipitation
1. Transfer the upper aqueous phase to a new tube containing isopropanol (500 µL of isopropanol per 750 µL of TRIzol used).
2. Mix by inverting 10-20 times.
3. Incubate for 10 min at RT.
4. Centrifuge at 12000 × g for 10 min at 4°C.
   At this step, the precipitated RNA will form a white pellet. The pellet size and level of visibility will depend on the RNA concentration.
5. Decant the supernatant, taking care not to disturb the pellet.
3. RNA washes
1. To the pellet, add 1 mL of 75% ethanol per 750 µL of TRIzol used.
2. Centrifuge at 7500 × g for 5 min at 4°C.
3. Decant the supernatant, taking care not to disturb the pellet.
4. Repeat steps 3a-c twice more.
5. Pulse spin at RT.
6. Carefully remove residual supernatant with a micropipettor, taking care not to disturb the pellet.
7. Air-dry the pellet at RT for 3-5 min.
8. Heat the tube uncapped at 65°C for 2-5 min.
4. RNA solubilization
1. Resuspend the pellet in 20-50 µl of water by pipetting up and down.
2. Incubate at 65°C for 2-5 min.
3. Vortex for 5-10 sec, pulse spin, and place on ice.
4. Measure the extracted total RNA concentration and purity.

The RNA is now ready for downstream applications or storage at -80°C for later use.

Reference

Toni LS, et al. (2018). Optimization of phenol-chloroform RNA extraction. MethodsX. 

**This is a suggested protocol and should be adjusted by the user accordingly**

For more info, visit our Methods of Virus Purificationpage.

While the above suggested protocol has been tested to be effective, if you are instead looking for a convenient solution for RNA isolation, complete with all buffers and reagents required, we recommend looking at the variety of viral & total RNA extraction kits that we have to offer: