RNA extraction is the initial step to isolate RNA molecules from biological samples. This process is necessary for various downstream applications, particularly quantitative Polymerase Chain Reaction (qPCR), which relies on highly purified RNA to accurately measure gene expression. The goal is to obtain RNA that is abundant and free from contaminants that could interfere with subsequent analyses. Without high-quality RNA, qPCR results would be significantly compromised.
The Fundamental Steps of RNA Extraction
RNA extraction involves sequential stages. Initially, cells or tissues undergo lysis, a process that disrupts their membranes to release intracellular contents, including RNA. Lysis buffers containing powerful denaturants, such as guanidine salts, are employed to break down cellular structures and inactivate RNA-degrading enzymes.
Following lysis, the released RNA must be separated from other cellular components like proteins, lipids, and DNA. This separation uses chemical and physical methods, partitioning RNA into an aqueous phase while proteins and DNA move into different phases or are retained by a matrix. Buffer and reagent conditions are controlled to optimize RNA recovery.
Subsequently, the separated RNA undergoes washing steps to remove residual contaminants or chemicals that could hinder downstream enzymatic reactions. These washes involve alcohol-based solutions, which purify RNA by removing impurities. Multiple washes ensure high purity.
The final stage is elution, where the purified RNA is recovered from the purification matrix or precipitated solution into a suitable, nuclease-free, buffer or water. This step makes the RNA readily available for direct use in sensitive molecular applications like reverse transcription and qPCR.
Key Considerations for High-Quality RNA
Achieving high-quality RNA is important for accurate qPCR results, requiring attention throughout the extraction process. One threat is RNase contamination; these ubiquitous enzymes degrade RNA, leading to fragmented samples. Maintaining an RNase-free environment through sterile, disposable plastics, dedicated workspaces, and RNase-free reagents is a common practice to prevent degradation.
Genomic DNA (gDNA) contamination also presents a challenge, as it can be co-purified with RNA and amplified during qPCR, leading to false positive results or overestimation of gene expression. To mitigate this, DNase treatment is integrated into the extraction workflow, either on-column during purification or as a separate solution-based step after initial RNA isolation. This enzymatic digestion targets and breaks down DNA, ensuring only RNA remains for analysis.
Other potential inhibitors, such as carryover salts, detergents, or phenol, can interfere with the enzymatic reactions of reverse transcription and PCR, reducing sensitivity and leading to inaccurate quantification. Adherence to washing protocols during extraction minimizes these inhibitory substances. Monitoring for inhibition can involve adding an exogenous RNA control to the reaction and observing its amplification.
After extraction, RNA quality assessment verifies its suitability for qPCR. Spectrophotometry is used to determine RNA concentration by measuring absorbance at 260 nm (A260) and to assess purity by calculating the A260/280 and A260/230 ratios. An A260/280 ratio between 1.8 and 2.0 indicates good purity, while an A260/230 ratio above 1.7 suggests minimal contamination from organic compounds or salts.
RNA integrity, which refers to the intactness of the RNA molecules, is assessed using techniques like gel electrophoresis or capillary electrophoresis systems such as the Agilent Bioanalyzer. The Bioanalyzer provides an RNA Integrity Number (RIN), a numerical score ranging from 1 (highly degraded) to 10 (intact), with scores of 7.0 or higher indicating RNA suitable for qPCR.
Common Approaches to RNA Extraction
Several methodologies are widely employed for RNA extraction, each leveraging different biochemical principles. Silica-based column extraction is a convenient method that relies on nucleic acids binding to a silica membrane in the presence of high concentrations of chaotropic salts. These salts disrupt water’s molecular structure and the silica matrix, creating conditions where RNA adheres to the membrane. Impurities are then washed away with alcohol-based buffers, and the purified RNA is eluted from the membrane using low-salt water or a buffer.
Organic extraction, exemplified by methods using TRIzol or TRI Reagent, involves a phase separation technique utilizing phenol and chloroform. Samples are homogenized in a monophasic solution containing phenol and guanidine isothiocyanate, which denatures proteins and maintains RNA integrity. Upon adding chloroform and centrifuging, the mixture separates into three distinct phases: an aqueous phase containing RNA, an interphase containing DNA, and an organic phase with proteins and lipids. The RNA-rich aqueous phase is then collected, and RNA is precipitated using isopropanol, followed by ethanol washes to remove residual contaminants.
Magnetic bead-based extraction offers another approach, utilizing microscopic magnetic particles coated with ligands that bind to nucleic acids under buffer conditions. After cell lysis, magnetic beads and a binding buffer are added to the sample, allowing RNA to adhere to the bead surfaces. An external magnetic field is then applied to pull the RNA-bound beads to the side of the tube, enabling the removal of unwanted cellular debris and contaminants through aspiration of the supernatant. Subsequent washing steps purify the RNA, and an elution buffer releases the RNA from the beads into a clean solution.