RNA extraction is a foundational technique in molecular biology, allowing isolation of ribonucleic acid from biological samples. This process is necessary to study gene expression, understand cellular processes, and diagnose diseases. TRIzol, a widely adopted chemical reagent, offers a reliable and efficient method for extracting high-quality RNA. This involves lysing cells, separating components, and purifying RNA for subsequent molecular analyses.
The TRIzol Reagent and Its Function
TRIzol is a monophasic solution for simultaneous isolation of RNA, DNA, and proteins from biological materials. Its primary components are guanidinium isothiocyanate and phenol, which work together for effective separation. This ready-to-use mixture rapidly dissolves biological material while maintaining RNA integrity.
Guanidinium isothiocyanate acts as a powerful chaotropic agent, disrupting cells and denaturing proteins, including RNases, which would otherwise degrade RNA. This denaturation releases nucleic acids for purification. Phenol, an organic solvent, aids in the separation of RNA from DNA and proteins during subsequent extraction steps. The acidic pH of TRIzol, around 5, ensures RNA remains in the aqueous phase, while DNA partitions into the interphase or organic phase.
The Step-by-Step Extraction Process
Homogenization
Homogenization involves lysing the biological sample within the TRIzol reagent. For tissue samples, mechanical disruption using a homogenizer is often required to break down cellular structure thoroughly. Cells in suspension can be lysed by repetitive pipetting directly in the TRIzol solution. Sample volume should not exceed 10% of the TRIzol volume, using 1 mL of TRIzol for every 50-100 mg of tissue or 5-10 x 10^6 animal cells. After mixing, the lysate should stand at room temperature for 5 minutes to ensure complete dissociation.
Phase Separation
After homogenization, add chloroform (0.2 mL of chloroform for every 1 mL of TRIzol reagent). Vigorously shake for 15 seconds to mix thoroughly, creating a milky appearance, then let sit at room temperature for 2-15 minutes. Centrifugation at 12,000 x g for 15 minutes at 4°C separates the mixture into three distinct layers. The colorless upper aqueous phase contains the RNA, DNA collects at the interphase, and proteins and lipids reside in the lower organic phase. Carefully transfer the upper aqueous phase to a new RNase-free tube, avoiding the interphase and organic layer.
RNA Precipitation
Precipitate RNA from the aqueous phase by adding isopropanol (0.5 mL of isopropanol per 1 mL of TRIzol used). Gently invert to mix and incubate at room temperature for 5-10 minutes, allowing RNA to form a pellet. Centrifuge (12,000 x g for 10 minutes at 4°C) to pellet the RNA as a translucent, gel-like pellet. Carefully discard the supernatant without disturbing the pellet.
RNA Washing
Wash the RNA pellet with 75% ethanol (1 mL of ethanol per 1 mL of TRIzol used) to remove salts and other impurities. Gently mix the pellet with ethanol by inversion or gentle pipetting, as vigorous vortexing can disperse it. Centrifuge (12,000 x g for 5 minutes at 4°C) to re-pellet the RNA. This washing step may be repeated to ensure maximum purity.
Resuspension
Carefully remove the ethanol supernatant and air-dry the RNA pellet for 5-10 minutes. Avoid over-drying, as this can make redissolving difficult. Dissolve the dried RNA pellet in 30-60 µL of RNase-free water or a 0.5% SDS solution. Gentle pipetting and incubation at 55-60°C for 10 minutes aid complete solubilization of the RNA.
Assessing RNA Purity and Yield
Assessing RNA purity and yield after extraction ensures suitability for downstream applications. UV spectrophotometry (e.g., NanoDrop) is a common method, measuring RNA sample absorbance at specific wavelengths.
The A260/A280 ratio evaluates protein contamination. Nucleic acids absorb light strongly at 260 nm, while proteins absorb at 280 nm. For pure RNA, an A260/A280 ratio of 1.9-2.1 is generally accepted. A lower ratio may indicate residual protein or phenol contamination.
The A260/A230 ratio indicates contamination from organic compounds, such as guanidinium salts, phenol, or carbohydrates. For pure RNA, this ratio commonly falls within 2.0-2.2. A significantly lower ratio suggests carryover of these contaminants from the extraction process.
Gel electrophoresis offers a visual assessment of RNA integrity and genomic DNA contamination. High-quality total RNA, when run on an agarose gel, should display distinct ribosomal RNA (rRNA) bands, typically two prominent bands for eukaryotic RNA (28S and 18S rRNA). Degraded RNA appears as a smear, indicating fragmentation rather than sharp bands. Genomic DNA contamination is visible as a high molecular weight band above the rRNA bands.
Safety Protocols and Handling
TRIzol and its associated chemicals are hazardous and require strict safety protocols. Phenol, a component of TRIzol, is corrosive to skin and eyes and toxic if inhaled, ingested, or absorbed dermally. Chloroform is also harmful, being carcinogenic and toxic by inhalation and ingestion.
Personal protective equipment (PPE) is necessary for all steps. Wear a lab coat, chemical-resistant gloves (e.g., nitrile), and safety glasses to protect against splashes and direct contact. Change gloves frequently, especially if any chemical contact occurs, as phenol can reduce nitrile glove efficacy over time.
All procedures involving TRIzol and chloroform must be performed in a certified chemical fume hood to ensure adequate ventilation and prevent inhalation of hazardous vapors. Biosafety cabinets do not provide sufficient respiratory protection for chemical fumes.
Proper waste disposal is essential. Liquid waste containing phenol and chloroform must be collected in designated hazardous waste containers. Contaminated solid waste, such as pipette tips and tubes, should be disposed of in appropriate hazardous waste receptacles. Never mix TRIzol waste with bleach or strong acids, as guanidinium thiocyanate can react to form highly toxic gases.
Common Issues and Troubleshooting
Common issues during TRIzol RNA extraction can impact yield or purity.
Low RNA yield often results from incomplete homogenization of the sample or accidental loss of the RNA pellet during washing steps. Ensuring thorough homogenization and careful pipetting when removing supernatants can help improve recovery. For samples with very low RNA content, adding a carrier like glycogen can make the pellet more visible and easier to handle.
Genomic DNA contamination is another common issue, often appearing as a viscous pellet or high molecular weight smear on an agarose gel. This can occur if the sample is not adequately homogenized, leading to insufficient shearing of the genomic DNA. Careful removal of only the aqueous phase during phase separation, avoiding the interphase where DNA resides, helps minimize this problem. A DNase treatment step after RNA precipitation can effectively remove residual genomic DNA.
Poor A260/A280 or A260/A230 ratios indicate contamination by proteins or residual reagents. A low A260/A280 ratio suggests protein or phenol contamination. This can be addressed by ensuring complete phase separation and performing an additional ethanol wash of the RNA pellet to remove remaining impurities. A low A260/A230 ratio often points to carryover of guanidine salts or other organic compounds from the extraction buffers. Increasing the number of 75% ethanol washes can help clear these contaminants from the purified RNA.