Complementary DNA, or cDNA, is a synthetic DNA molecule created from an RNA template through a process called reverse transcription. This conversion is a fundamental step in many molecular biology applications, allowing researchers to study gene expression by analyzing the protein-coding sequences of genes. cDNA is widely used in cloning, gene expression analysis, and RNA sequencing, providing a stable form of genetic information derived from the more fragile RNA molecules.
Factors Influencing cDNA Stability
cDNA, despite being more stable than its RNA precursor, is still susceptible to degradation through several mechanisms. One significant factor is enzymatic degradation, primarily caused by nucleases like DNases. These enzymes, which break down DNA, can be introduced through improper handling, contaminated reagents, or non-sterile equipment, leading to the breakdown of the cDNA molecule.
Chemical degradation also poses a threat to cDNA integrity. Hydrolysis, the breakdown of molecules by water, can occur over time, especially at higher temperatures. Extreme pH conditions, both highly acidic and highly alkaline environments, can damage DNA by disrupting its chemical bonds and backbone structure.
Physical stress, such as repeated freeze-thaw cycles, can significantly impact cDNA integrity. Each cycle of freezing and thawing can cause the formation of ice crystals, which can physically shear and fragment the DNA strands. This mechanical stress can lead to strand breaks and a reduction in the average size of the cDNA molecules, potentially affecting downstream applications. Higher temperatures accelerate chemical and enzymatic activity, contributing to faster degradation.
Optimal cDNA Storage Conditions
The stability of cDNA varies significantly depending on storage temperature and conditions. For short-term use, cDNA can be stored at 4°C, typically remaining stable for a few days to a week. It can remain stable for up to three weeks at 4°C. However, this temperature is generally not recommended for storage beyond immediate experimental needs.
For mid-term storage, -20°C is a common temperature that can preserve cDNA for several months to about a year. It is important to avoid using frost-free freezers for -20°C storage, as their fluctuating temperatures can induce repeated freeze-thaw cycles, compromising sample integrity. Long-term storage, spanning multiple years, requires ultra-low temperatures, with -80°C being the gold standard.
Proper handling and storage buffers are crucial for maintaining cDNA stability. Aliquoting, or dividing the cDNA into smaller, single-use portions, is a widely recommended practice to minimize the number of freeze-thaw cycles any given sample undergoes. Storing cDNA in nuclease-free water or, preferably, in a buffer such as Tris-EDTA (TE buffer) is also important. TE buffer helps maintain a stable pH, and its EDTA component chelates metal ions that can activate nucleases, thus preventing enzymatic degradation. Using nuclease-free reagents and sterile techniques throughout the cDNA synthesis and storage process is important to prevent contamination and subsequent degradation.
Assessing cDNA Integrity
Verifying the quality of stored cDNA is an important step before using it in sensitive molecular biology applications. Quantitative measurements provide information on the amount and purity of the cDNA. Spectrophotometry measures the absorbance of light at specific wavelengths (A260 and A280 nm) to estimate concentration and purity, indicated by the A260/A280 ratio. However, this method can be influenced by contaminants that also absorb UV light.
More accurate quantification can be achieved using fluorometric methods. These methods utilize fluorescent dyes that bind specifically to double-stranded DNA, providing a more precise measurement of cDNA concentration even in the presence of contaminants. The fluorescent signal is directly proportional to the amount of nucleic acid present, offering higher sensitivity than absorbance-based techniques.
Qualitative assessment methods visualize the integrity of the cDNA. Gel electrophoresis can be used to check for degradation; intact cDNA typically appears as a smear representing a range of lengths, while degraded samples may show a shift towards smaller fragments or a more diffuse smear. However, this method is not sensitive enough to detect subtle degradation or provide quantitative data. The most reliable test for cDNA integrity is its performance in downstream functional assays, such as polymerase chain reaction (PCR) or quantitative PCR (qPCR). If the cDNA yields expected and consistent results in these applications, it confirms its suitability for the intended experimental purpose.