Activity 4-1 - RNA Extraction via Guanidine Thiocyanate and Phenol

RNA Extraction

RNA extraction is a fundamental technique in molecular biology that allows scientists to isolate RNA from cells for downstream applications like RT-qPCR, RNA sequencing, or gene expression analysis. RNA must be extracted carefully because it is prone to degradation by RNases—enzymes present both inside and outside cells. There's a misconception that one may use RIPA buffer, which is an excellent reagent for protein extraction. However, it lacks the strong chaotropic and phase-separation properties needed for clean RNA isolation. Also, it doesn’t inactivate RNases as effectively as RNA-Bee. This is why RIPA is avoided in RNA work—it may allow RNA degradation.

To extract RNA efficiently, cells are disrupted using a powerful reagent like RNA-Bee, which contains phenol and guanidine thiocyanate. Guanidine thiocyanate is a chaotropic salt, which breaks down protein structures and denatures enzymes (including RNases), preserving the integrity of the RNA. Phenol plays a different but equally important role. It's nonpolar, meaning it does not mix well with polar substances like RNA. When mixed with cell lysates, phenol helps separate the polar nucleic acids (RNA and DNA) from nonpolar molecules such as lipids and proteins. As a result, nucleic acids stay in the aqueous (top) layer, while proteins and lipids settle in the organic (bottom) layer. Think of phenol as oil in a salad dressing—it helps separate water-loving (hydrophilic) ingredients from fat-loving (hydrophobic) ones.

Once cells are lysed in RNA-Bee, chloroform is added. Chloroform is another nonpolar solvent, and it mixes with phenol to form a denser organic layer. When centrifuged, the mixture separates into three layers. Imagine shaking a bottle of oil, vinegar, and honey. Eventually, the heaviest (honey) settles at the bottom, and the lightest (vinegar) floats to the top. This clean separation is crucial so you can carefully collect the top layer (aqueous phase) that contains pure RNA.

• Aqueous (top) – contains RNA.
• Interphase (middle) – DNA and some proteins.
• Organic (bottom) – phenol, chloroform, denatured proteins, and lipids.

The next step is RNA precipitation, where isopropanol (a type of alcohol) is added to the aqueous phase. RNA is less soluble in alcohol, especially at cold temperatures, so it comes out of solution and forms a visible pellet after centrifugation. To maximize precipitation, the sample is usually incubated overnight at 4°C, which helps RNA clump together into a pellet you can spin down easily.

Even after RNA is precipitated with isopropanol, some salts and impurities might remain. To clean the pellet further, 100% ethanol is added. Ethanol also helps remove any residual isopropanol, which can interfere with downstream reactions such as reverse transcription or PCR. However, if 100% ethanol is not available, 95% ethanol containing methanol can be used as an alternative for the wash step. While slightly less efficient due to its water content, 95% ethanol is still effective at removing salts and impurities. The small amount of methanol present should not pose a problem as long as the RNA pellet is thoroughly air-dried before resuspension. For best results, an additional ethanol wash can be included to enhance RNA purity when using 95% ethanol, and care should be taken to allow complete evaporation of any residual solvent.

Once dry, the purified RNA is resuspended in RNA-free water. You can measure its concentration using a Nanodrop spectrophotometer, which detects RNA at 260 nm. The purity of the RNA can be assessed by the A260/A280 ratio. An ideal A260/A280 ratio for pure RNA is typically around 2.0. This indicates minimal contamination from proteins, phenol, or other organic compounds, which tend to absorb more strongly at 280 nm. If the ratio is significantly lower than 2.0 (e.g., 1.6–1.8), it suggests protein contamination or incomplete removal of phenol. In such cases, you may want to repeat the ethanol wash or perform a column-based cleanup for higher purity. Additionally, you can check the A260/A230 ratio, which ideally should be 2.0–2.2. Lower values here may indicate contamination by salts, carbohydrates, or residual guanidine from the RNA-Bee reagent. Ensuring both ratios are within ideal ranges will give you the best results in sensitive downstream applications like RT-PCR, qPCR, or RNA-seq. Once quantified and verified, your RNA is ready to use or can be stored long-term at –80°C to preserve its integrity.

Protocol for RNA Extraction