Complementary DNA, or cDNA, is a stable, double-stranded DNA molecule that serves as a copy of a fragile messenger RNA (mRNA) template. This conversion, performed in the laboratory using an enzyme called reverse transcriptase, removes non-coding regions, making the resulting DNA molecule easier to work with for applications like gene expression analysis, sequencing, and Polymerase Chain Reaction (PCR). Since the integrity of this molecule directly impacts the reliability of all subsequent experiments, establishing a meticulous storage protocol is paramount for maintaining the quality and usability of the sample over time.
Essential Steps Before Storage
The first step in preserving cDNA involves immediate preparation after the reverse transcription reaction to maximize its long-term stability. A crucial decision is the choice of storage medium, which involves a trade-off between long-term chemical stability and compatibility with downstream enzymatic reactions. Nuclease-free water is often preferred when the sample will be used almost exclusively for PCR, as the water minimizes the chance of reaction inhibition.
For long-term preservation, a low-concentration Tris-EDTA (TE) buffer is considered superior. The Tris component provides pH buffering, protecting the DNA from acid hydrolysis that occurs in unbuffered water. EDTA chelates divalent cations like magnesium, which are necessary cofactors for DNase enzymes, thereby inhibiting degradation. This buffer stabilizes the sample over months or years, making it the favored choice for archival storage.
Before any sample is placed into a freezer, precise quantification is necessary to ensure experimental reproducibility across different assays and time points. Methods like UV spectrophotometry (e.g., Nanodrop) or fluorescent dye-based assays (e.g., Qubit) provide an accurate measurement of the cDNA concentration. Knowing the exact concentration prevents the need to thaw the main stock simply to re-quantify, which is one of the primary causes of sample degradation.
The most important step preceding long-term storage is dividing the sample into smaller, single-use aliquots. This practice directly addresses the major threat to nucleic acid integrity: repeated freeze-thaw cycles. By creating multiple small volumes, only the necessary amount is thawed for a single experiment, leaving the bulk of the sample securely frozen. The aliquots should be stored in sterile, clearly labeled microcentrifuge tubes or cryogenic vials that are designed to remain securely sealed and withstand ultra-low temperatures without cracking.
Temperature Regimes for cDNA Storage
The longevity of cDNA is directly correlated with the temperature at which it is maintained, where lower temperatures significantly reduce the rate of chemical and enzymatic degradation. For true long-term storage, defined as preserving the sample for many months or years, the ultra-low temperature of $-80^\circ\text{C}$ is the established gold standard. At this temperature, molecular motion is drastically reduced, effectively halting the chemical processes that lead to spontaneous strand breakage and degradation.
Maintaining samples at $-80^\circ\text{C}$ requires a dedicated, reliable ultra-low temperature freezer that can recover quickly from door openings and maintain a consistent temperature. This environment minimizes the risk of damage, ensuring the cDNA remains intact for future applications. Samples stored under these conditions are stable for indefinite periods, provided the freezer remains operational.
Intermediate storage, such as at $-20^\circ\text{C}$, is acceptable for shorter durations, typically ranging from a few weeks to a few months. While the lower temperature provides better stability than refrigeration, it does not completely stop all chemical activity. For highly valuable or low-abundance cDNA transcripts, researchers should avoid relying on $-20^\circ\text{C}$ for storage extending beyond a few months to prevent subtle, progressive degradation that could affect quantitative results.
Refrigerated storage at $4^\circ\text{C}$ should be used only for immediate, short-term needs, such as holding a sample overnight or for a few hours before a planned experiment. This temperature is insufficient for actual archival storage, as the rate of chemical and potential enzymatic degradation remains too high. Any sample stored at $4^\circ\text{C}$ for more than a day or two risks a noticeable decline in quality.
Preventing Degradation and Contamination
Protecting cDNA involves maintaining a consistent temperature and controlling handling practices. The single most destructive action is repeated freezing and thawing, which is why aliquoting is mandatory. Each cycle causes physical shear forces as ice crystals form and melt, leading to DNA fragmentation. When an aliquot is needed, it should be thawed quickly on ice and returned to the freezer immediately, minimizing the time the sample spends in the liquid state where degradation is active.
Contamination by nucleases—enzymes that break down nucleic acids—presents a significant biological threat. Since DNases are ubiquitous and can rapidly destroy a sample, all materials coming into contact with the cDNA must be certified nuclease-free. This requires using dedicated reagents, sterile plasticware, and maintaining a meticulous technique, including the use of fresh gloves at all times to prevent the introduction of enzymes from the skin or environment.
Maintaining a precise inventory is an organizational action that directly supports sample integrity by preventing unnecessary thawing. A detailed inventory system should track the freezer location, the date of synthesis, the quantified concentration, and the number of remaining aliquots for each cDNA stock. By logging every use, researchers can easily locate the correct aliquot without having to retrieve and examine the main stock.