Phenol Chloroform RNA Extraction: A Reliable Approach

Extracting high-quality RNA is essential for molecular biology research, as degraded or impure samples can compromise results. Phenol-chloroform extraction remains widely used for its efficiency in separating RNA from DNA and proteins while preserving RNA integrity.

Despite the availability of commercial kits, many researchers rely on this cost-effective and reliable technique. Understanding key steps and factors influencing RNA yield and purity ensures successful extractions.

Molecular Principle

Phenol-chloroform RNA extraction exploits the differing solubilities of nucleic acids and proteins in aqueous and organic phases. When a biological sample is mixed with phenol-chloroform, phase separation occurs. RNA remains in the upper aqueous phase due to its hydrophilic nature, while proteins and lipids partition into the organic phase or interphase. Phenol and chloroform disrupt hydrogen bonding and denature proteins, preventing contamination of the RNA fraction.

In acidic conditions, phenol retains DNA in the organic phase while RNA stays in the aqueous layer. This pH-dependent behavior minimizes DNA contamination, which can interfere with downstream applications like quantitative PCR and RNA sequencing. Chloroform enhances phase separation by increasing the density of the organic layer, reducing cross-contamination.

Isoamyl alcohol is often included to reduce foaming and improve phase clarity. Without it, emulsions can form, making it difficult to recover the aqueous phase cleanly. The optimized 25:24:1 ratio of phenol, chloroform, and isoamyl alcohol maximizes RNA yield while minimizing protein carryover.

Key Components And Solutions

The composition and quality of reagents directly influence RNA yield and purity. Phenol disrupts hydrogen bonds and denatures proteins, effectively separating them from nucleic acids. Acidic phenol (pH ~4.5) drives DNA into the organic phase while keeping RNA in the aqueous layer. Commercial phenol solutions are often stabilized with antioxidants like 8-hydroxyquinoline to prevent oxidation, which could degrade RNA.

Chloroform enhances phase separation and clarifies the interface between the aqueous and organic layers. The 25:24 phenol-to-chloroform ratio optimizes RNA recovery while minimizing emulsification. Isoamyl alcohol, included at a 1:25 ratio relative to phenol, suppresses foaming and stabilizes the interface.

Aqueous buffers also impact RNA integrity. Sodium acetate (0.3 M, pH 5.2) promotes RNA precipitation by neutralizing the phosphate backbone’s negative charge. Ethanol or isopropanol facilitates RNA precipitation, with ethanol providing higher purity and isopropanol offering greater recovery efficiency. Glycogen or linear polyacrylamide improves precipitation, particularly for low-yield samples.

Standard Procedure

RNA extraction begins with tissue or cell lysis, where samples are homogenized in a chaotropic solution containing phenol to disrupt cellular structures. Mechanical disruption, such as bead milling or rotor-stator homogenization, is often necessary for fibrous tissues. Enzymatic digestion with proteinase K may be used for samples with high protein content. Keeping samples cold, typically on ice or at 4°C, helps preserve RNA integrity by inhibiting RNases.

After homogenization, chloroform is added to induce phase separation. Vortexing or inversion ensures uniform solvent distribution. Centrifugation at 12,000 to 16,000 × g for 15 minutes at 4°C yields distinct layers: the upper aqueous phase containing RNA, the interphase trapping DNA and proteins, and the lower organic phase sequestering lipids. Careful pipetting prevents contamination of the RNA fraction.

RNA is precipitated from the aqueous phase using alcohol, typically isopropanol or ethanol, combined with a salt like sodium acetate or lithium chloride. Incubation at -20°C or -80°C for at least 30 minutes enhances precipitation, especially for low-yield samples. Centrifugation at 12,000 × g for 10–15 minutes forms an RNA pellet, which is washed with 70% ethanol to remove residual salts and phenol. Proper drying before resuspension prevents ethanol carryover, which can inhibit enzymatic reactions.

Importance Of pH Control

The pH of the extraction solution determines the selective separation of nucleic acids. Acidic conditions (pH ~4.5) drive DNA into the organic phase while keeping RNA in the aqueous layer. DNA becomes protonated and hydrophobic at lower pH, causing it to partition into the phenol-chloroform phase, while RNA remains hydrophilic. Without proper pH control, DNA contamination can persist, interfering with RNA sequencing and RT-qPCR.

This is particularly important for DNA-rich samples, such as those from nuclear-dense tissues. Even slight deviations from the optimal range can lead to DNA retention in the aqueous phase, complicating RNA purity. Commercial phenol solutions are often pre-equilibrated, but their stability degrades over time due to oxidation, necessitating regular pH checks with pH paper or a calibrated meter.

Handling And Storage

Strict precautions are necessary to prevent RNA degradation by RNases. Laboratory surfaces, pipettes, and glassware should be treated with RNase inhibitors or baked at 180°C to inactivate enzymes. Using RNase-free plasticware and filter tips further reduces contamination risks. Gloves should be worn and changed frequently to prevent cross-contamination. Maintaining a dedicated workspace for RNA work helps preserve sample integrity.

Extracted RNA must be stored properly to prevent hydrolysis and oxidation. Short-term storage at -80°C in nuclease-free water or TE buffer (pH 7.0–8.0) is recommended. For long-term preservation, RNA can be suspended in ethanol or stored as a precipitate at -80°C, which is more effective than aqueous solutions. Lyophilization is another option, as it removes moisture that could facilitate degradation. Avoiding repeated freeze-thaw cycles prevents RNA fragmentation. Aliquoting RNA into smaller volumes minimizes the need for repeated thawing, preserving sample quality.

Assessing RNA Purity

Evaluating RNA purity is critical for reliable downstream applications. Contaminants like proteins, phenol residues, and genomic DNA can interfere with enzymatic reactions and bias results. Spectrophotometric analysis using a NanoDrop or similar device provides a quick assessment. The A260/A280 ratio indicates protein contamination, with values between 1.8 and 2.1 reflecting high purity. A lower ratio suggests protein carryover, which can be remedied with additional purification steps like ethanol precipitation. The A260/A230 ratio assesses contamination from phenol, salts, or organic compounds, with values above 2.0 indicating minimal interference.

Capillary electrophoresis systems like the Agilent Bioanalyzer or Fragment Analyzer provide RNA integrity numbers (RIN) that quantify degradation. A RIN above 7 is suitable for most applications, while values below 5 indicate significant degradation, which may impact transcriptome analysis. Agarose gel electrophoresis offers a visual confirmation of RNA quality, with intact samples displaying distinct 28S and 18S ribosomal RNA bands in a 2:1 ratio. If degradation is observed, potential causes include RNase contamination, improper storage, or excessive mechanical shearing during extraction. Addressing these issues early prevents setbacks in gene expression studies and sequencing projects.