Complementary DNA, or cDNA, is a synthetic DNA molecule created from an RNA template using an enzyme called reverse transcriptase. Unlike genomic DNA, which contains both coding and non-coding regions, cDNA includes only the coding sequences that instruct cells to produce proteins. This makes cDNA a valuable tool in various biological applications, such as gene cloning, gene expression analysis, and diagnostics. Maintaining the integrity and usability of cDNA through proper storage is important for the success of future experiments and analyses.
Factors Influencing cDNA Stability
Several factors can compromise the integrity of cDNA over time, leading to degradation. Enzymes known as nucleases are a major threat, as they break down nucleic acids by cleaving bonds between nucleotides. These enzymes require certain metal ions to function, making their presence particularly damaging to DNA samples. Extreme pH levels also pose a risk; DNA molecules are most stable within a neutral pH range, typically between 5 and 9. Both highly acidic (pH 5 or lower) and highly alkaline (pH 9 or higher) conditions can destabilize DNA, leading to the loss of bases or denaturation.
Temperature fluctuations, especially repeated cycles of freezing and thawing, can also degrade cDNA. When samples freeze, water expands, which can physically stress and potentially break DNA molecules. Additionally, contaminants like bacteria or fungi can introduce their own nucleases, accelerating cDNA degradation. Maintaining a stable environment is therefore important to protect cDNA from these various detrimental influences.
Optimal Storage Conditions
Storing cDNA at appropriate temperatures is important for its long-term preservation. For short-term storage, typically a few weeks, cDNA can be kept at -20°C. For long-term preservation, extending to months or years, a temperature of -80°C or colder is recommended to minimize degradation. These very low temperatures significantly reduce molecular activity, effectively halting the chemical and enzymatic processes that cause DNA breakdown.
The choice of storage buffer contributes to cDNA stability. Tris-EDTA (TE) buffer is widely used because its components protect the nucleic acid. Tris maintains a stable pH, typically around 8.0, which is suitable for DNA stability. EDTA acts as a chelating agent, binding to metal ions like magnesium that are necessary for nuclease activity. By sequestering these ions, EDTA inactivates nucleases, preventing them from degrading the cDNA.
Appropriate storage containers are also important for preservation. Using sterile, low-binding, screw-cap vials is advisable. Low-binding tubes minimize the adherence of small amounts of cDNA to the plastic, ensuring more of the sample remains in solution. Screw-cap vials provide a secure seal, preventing evaporation and reducing contamination.
Handling Practices for Preservation
Proper handling practices are important to maintain cDNA integrity during storage and retrieval. Aliquoting, the process of dividing a larger sample into multiple smaller portions, is an important practice. This minimizes the number of times the entire stock solution is exposed to freeze-thaw cycles. Each small aliquot can be thawed and used as needed, leaving the remaining stock undisturbed in its frozen state.
Maintaining a sterile environment is also important to prevent contamination. Using sterile tubes, pipette tips, and working in a clean area reduces the introduction of microorganisms that could produce nucleases. Contamination can lead to rapid degradation of cDNA, compromising experimental results. Proper labeling of each tube with details like concentration, date, and project name helps in sample management and avoids confusion.
When retrieving frozen cDNA, allowing samples to thaw gently on ice is recommended. Rapid thawing at room temperature or with heat can induce localized temperature and pH shifts that may contribute to degradation. Gentle thawing helps to maintain the sample’s stability and prevent localized damage to the DNA molecules.
Assessing Stored cDNA Quality
Verifying the integrity and quantity of cDNA after storage is important for successful downstream applications. Spectrophotometry is a method used to measure the concentration and purity of a DNA sample. This technique measures the amount of ultraviolet light absorbed by the DNA at a specific wavelength (260 nm) to determine its concentration. The ratio of absorbance at 260 nm to 280 nm (A260/A280 ratio) indicates purity, with a ratio between 1.7 and 2.0 considered pure for DNA; deviations can suggest contamination by proteins or other substances.
Gel electrophoresis provides a visual assessment of cDNA integrity. In this method, cDNA fragments are separated by size as they migrate through a gel matrix under an electric field. Intact cDNA appears as a distinct band at the expected size, while degraded cDNA shows a smear or multiple smaller bands, indicating fragmentation. This technique qualitatively measures if the cDNA has maintained its structural integrity.
Quantitative PCR (qPCR) serves as a functional assay to confirm the cDNA’s suitability for amplification. This method assesses whether the stored cDNA can still be effectively amplified and detected, providing insight into its functional quality for molecular biology experiments. These checks confirm storage methods were effective and that the cDNA remains suitable for its intended use.