The Role of Centrifugation in High-Purity DNA, RNA and Protein Extraction

Centrifugation Fundamentals in Nucleic Acid Extraction

Centrifugation Steps in DNA and RNA Extraction

The extraction of DNA and RNA is a delicate process that hinges on effectively separating nucleic acids from cellular components. Centrifugation is indispensable in this process, which involves a series of steps, each designed to refine the purity of the nucleic acids.

Cell Lysis: The journey begins with the disruption of the cell membrane, a process that can be achieved through various methods such as chemical, enzymatic, or mechanical means. Following lysis, centrifugation comes into play, spinning the sample to sediment cellular debris and leaving nucleic acids in the supernatant. The centrifugation conditions are meticulously set to ensure the nucleic acids remain in the liquid phase while the denser cellular fragments are compacted into a pellet.

Removal of Proteins and Other Contaminants: Substances such as a binding solution or a phenol-chloroform mixture are introduced at this stage. Subsequent centrifugation forms distinct layers: an organic phase, an interphase, and an aqueous phase, with the latter housing the DNA or RNA. Precise centrifugation parameters are essential here to ensure a clear demarcation between these phases, facilitating the careful extraction of the nucleic acid-rich aqueous layer.

Precipitation of Nucleic Acids: Adding alcohol and salt to the aqueous phase induces the precipitation of DNA or RNA. A centrifugal force is then applied to collect the nucleic acids into a pellet. The centrifugation speed and duration are pivotal in determining the purity and the quantity of the nucleic acids harvested at this stage.

Washing: The nucleic acid pellet is washed with a cold alcohol solution to remove any lingering salts and solvents. Centrifugation follows, allowing the nucleic acids to settle once more into a pellet. The supernatant is then discarded, carefully considering the centrifuge's speed to prevent disturbing the fragile pellet.

Redissolving Nucleic Acids: After drying, the pellet is resuspended in a suitable buffer or water. While centrifugation is not actively involved in this step, a brief spin can help gather any droplets clinging to the tube walls, aiding in the pallet’s dissolution.

Further Purification (Optional): For additional purity or concentration, centrifugation can be employed using a micro-concentrator column. The sample is loaded onto the column and spun, allowing the nucleic acids to adhere to the matrix while impurities are washed away. A final spin with buffer or water elutes the purified nucleic acids.

Quality Control (Optional): After extraction, centrifugation can be instrumental in preparing samples for quality and concentration assessments. A quick spin before loading onto gels or other analytical instruments ensures the removal of any particulates that could skew results.

Throughout these stages, the centrifugation conditions—such as relative centrifugal force (RCF), time, and temperature—are carefully managed to suit the sample type and the desired nucleic acid purity. These parameters are vital for effectively separating target molecules from undesirable elements.

Common Approaches to DNA, RNA, and Protein Isolation

Centrifugation is a cornerstone technique in the laboratory, particularly crucial for extracting DNA, RNA, and proteins.

Protocols for the isolation of DNA or RNA initially depend on the source of isolation. Whether it’s fresh host tissue, formalin-fixed paraffin-embedded tissue (FFPE), cells isolated from tissue culture, or E. coli fermentation, its initial steps for isolation vary and may include homogenization, retrieval of the target tissue in the case of FFPE, and centrifugation.(1,2)

Guanidinium thiocyanate-phenol-chloroform.

This method is one of the most widely used for isolating nucleic acids and proteins from homogenized tissues and pelleted cells. This chaotropic agent (something capable of disrupting hydrogen bonds in a solution) allows the lysis of cells and the degradation of proteins for DNA, RNA, and protein extraction. The reaction is performed at a reduced pH, and the addition of chloroform, followed by centrifugation, creates an aqueous phase containing RNA, an interphase, and an organic phase containing proteins and lipids. The isolation of RNA from the aqueous phase is conducted via isopropyl alcohol precipitation, and multiple washes with ethanol have been reported to improve yields compared to the standard single wash. (3) The isolation of DNA and proteins from the interphase and organic phase can be performed by directly adding ethanol and pelleting the DNA via centrifugation. The DNA pellet is further processed and washed with sodium citrate/ethanol solutions and then solubilized with sodium hydroxide or a base before titrating the pH with HEPES. The proteins are found within the supernatant from the centrifuged interphase and organic phase layers, which resulted in the pelleting of the DNA. This entire process is achieved by incubating the supernatant with isopropanol followed by centrifugation to pellet the proteins. The subsequent steps for protein washes of the pellet are conducted using a 0.3 M guanidine hydrochloride solution in 95% ethanol. The pellet is then re-suspended in Sodium Dodecyl Sulfate (SDS) and placed at 50°C on a heat block or in a water bath before the final centrifugation to remove insoluble products. (2)

Alkaline Extraction

Alkaline extraction methods are most commonly used for isolating plasmid DNA from E. coli cell strains. This technique is achieved by using a series of alkaline solutions to chelate ions and maintain osmolarity. Following this procedure, an SDS and sodium hydroxide-containing solution is utilized for cell lysing and denaturing the internal genomic cell DNA without harming the plasmid DNA, which cannot be linearized due to its covalently closed nature. Finally, the reaction is neutralized with a potassium acetate and acetic acid solution, allowing the plasmid DNA to renature without collecting the genomic DNA, thereby preventing contamination of the plasmid preparation. (2)

Solid-Phase Extraction

These extraction methods utilize materials that can bind or undergo adsorption processes via principles of hydrogen bonding, ion exchange, affinity, or size exclusion. This procedure offers a rapid method of sample isolation, utilizing specially designed columns that employ centrifugal forces to purify samples. The materials range from silica to diatomaceous earth (DE), which contains large percentages of silica, glass particles, or magnetic beads.(2)

Optimal Centrifugation Conditions for Nucleic Acid Purity

Achieving high-purity DNA and RNA is paramount for their use in sensitive downstream applications. Therefore, the centrifugation conditions during extraction are not arbitrary but carefully calibrated to enhance the quality of the nucleic acids.

Relative Centrifugal Force (RCF): The RCF is pivotal for sedimenting particles without damaging the delicate nucleic acids. The optimal RCF is sample-specific; for instance, bacterial cells often require a more robust force than mammalian cells.

Spin Time: The duration of each centrifugal step is critical for complete separation. Insufficient spin time may leave the separation incomplete, while excessively long spins risk pulling down impurities alongside the desired nucleic acids.

Temperature: To protect especially fragile RNA from enzymatic degradation, centrifugation is often performed at reduced temperatures, such as 4°C. Additionally, certain precipitation reactions are more efficient at lower temperatures, further enhancing the extraction process.

Rotor Type: The centrifuge rotor's design affects sedimentation speed and path length. Fixed-angle rotors are typically used for pelleting due to their shorter path length, while swing-out rotors are preferred for phase separation due to their horizontal orientation during spinning.

Acceleration and Deceleration Rates: The rate at which the centrifuge reaches and reduces speed can be crucial. Rapid acceleration can hasten pelleting, but a measured acceleration is necessary for samples requiring gentle handling or when dealing with gradient layers. Deceleration rates are equally important, as they can influence whether a soft pellet is disturbed.

Speed, Time, and Temperature Control

Navigating the intricacies of centrifugation requires a delicate balance of speed, duration, and thermal conditions. Each element is a cornerstone in safeguarding the integrity and purity of DNA and RNA samples.

Speed: The centrifugal force, quantified in revolutions per minute (rpm) or relative centrifugal force (RCF), is tailored to the specific gravity and dimensions of the target molecules. While a robust force is necessary to sediment nucleic acids effectively, an overly vigorous approach can fragment long DNA chains or inadvertently mix the desired nucleic acids with other cellular constituents. Although established protocols provide a baseline, adjustments may be warranted to accommodate unique sample attributes or experimental demands.

Time: The centrifugation period is equally pivotal. An abbreviated spin might fail to segregate contaminants adequately, whereas an excessively long cycle could compact the pellet too tightly, complicating its resuspension or potentially harming the nucleic acids. The ideal centrifugation duration hinges on the interplay between the applied speed and the sample's characteristics.

Temperature Control: Temperature's role in maintaining nucleic acid stability cannot be overstated, particularly when handling RNA or samples prone to nuclease activity. Centrifuges with precise temperature regulation are invaluable, maintaining conditions as low as 4°C or adhering to protocol-specific refrigerated settings. This vigilance in temperature management safeguards sensitive nucleic acids against degradation.

By deftly adjusting these three parameters, scientists can refine the centrifugation process, enhancing the yield and the purity of the nucleic acids extracted. While general protocols serve as a reference, tailoring these conditions to each distinct scenario is often necessary. Advanced centrifuges that offer meticulous control over these variables are instrumental in bolstering the consistency and effectiveness of nucleic acid extraction methodologies.

Sources

1. Miskimen KLS, Miron PL. Isolation of Genomic DNA from Mammalian Cells and Fixed Tissue. Curr Protoc. 2023 Jun;3(7):e818.
2. Tan SC, Yiap BC. DNA, RNA, and protein extraction: the past and the present. J Biomed Biotechnol. 2009;2009:574398.
3. Toni LS, Garcia AM, Jeffrey DA, Jiang X, Stauffer BL, Miyamoto SD, et al. Optimization of phenol-chloroform RNA extraction. MethodsX. 2018 May 30;5:599–608.