A Cells-to-CT kit is a specialized tool used in molecular biology laboratories to streamline the process of preparing biological samples for gene expression analysis. This kit simplifies a multi-step procedure by allowing researchers to move directly from cultured cells to the detection of specific gene activity. Its primary goal is to make gene expression analysis faster and more accessible. It eliminates time-consuming and labor-intensive steps, making it useful for high-throughput experiments.
What is a Cells to CT Kit?
A Cells-to-CT kit prepares cell lysates for reverse transcription quantitative polymerase chain reaction (RT-qPCR), a common method for measuring gene expression. Instead of lengthy RNA purification, these kits directly convert messenger RNA (mRNA) from cells into complementary DNA (cDNA). This direct approach bypasses columns, heating, or centrifugation associated with RNA isolation, addressing challenges like RNA degradation and sample loss.
The kit simplifies sample preparation from cultured cells, handling 10 to 100,000 cells per reaction. This direct cell-to-cDNA approach significantly reduces preparation time, often to under ten minutes. This streamlined workflow minimizes contamination and human error, leading to consistent and reliable results for gene expression studies.
The Simple Process: How the Kit Works
Using a Cells-to-CT kit involves key stages occurring sequentially within a single tube or well, minimizing sample transfers. Cultured cells are first washed with a solution like phosphate-buffered saline (PBS), then mixed with a specialized Lysis Solution. This solution breaks open cell membranes, releasing RNA, while inactivating RNases that could degrade it.
Following cell lysis, an optional step adds DNase I to the Lysis Solution, which degrades genomic DNA. This step ensures that only RNA-derived cDNA is amplified in downstream reactions, preventing false positives from genomic DNA contamination. After a brief incubation, a Stop Solution is added to the lysate. This solution neutralizes the lysis reagents, ensuring they do not interfere with subsequent enzymatic reactions.
Once prepared, the lysate is directly used for reverse transcription, converting mRNA into cDNA using an RT Enzyme Mix and RT Buffer. This cDNA is then ready for real-time PCR amplification, avoiding intermediate RNA purification steps. The entire sample preparation completes rapidly, often within 5 to 7 minutes, making it suitable for high-throughput applications using automated liquid handling systems.
Why Researchers Use Cells to CT Kits
Researchers widely adopt Cells-to-CT kits due to their advantages over traditional RNA extraction methods. A primary benefit is speed and reduced hands-on time, allowing preparation of many samples, such as 96, for RT-qPCR in under ten minutes. This rapid processing benefits large-scale experiments like RNA interference (RNAi) screening or evaluating gene expression changes.
The kits also minimize sample loss by eliminating multiple transfer and purification steps, which can lead to material loss, especially with small or precious samples. This workflow enhances consistency and reproducibility by reducing variability from complex manual procedures. Additionally, the process decreases plastic waste compared to traditional RNA purification kits, offering a more sustainable lab approach. These efficiencies make Cells-to-CT kits useful for gene expression analysis in drug development, biomarker discovery, and pathogen detection.
Understanding the “CT” in Cells to CT
The “CT” in Cells-to-CT stands for Cycle Threshold, a term central to quantitative Polymerase Chain Reaction (qPCR). In qPCR, a fluorescent signal is generated as DNA is amplified. The CT value represents the cycle number at which this signal crosses a predetermined threshold level, significantly above background noise. This threshold is set within the exponential phase of the amplification curve, where the reaction is most efficient and accurate.
The CT value provides a quantitative measure of the initial amount of target nucleic acid in the sample. A lower CT value indicates earlier detection of the fluorescent signal, meaning a higher initial concentration of the target gene. Conversely, a higher CT value suggests a lower initial amount. For instance, a CT value below 29 cycles indicates a high target concentration, while values above 38 cycles suggest a very low amount or potential reaction issues. Understanding these values is important for accurately interpreting gene expression levels and making meaningful comparisons between different samples.