Cells-to-CT™ kits provide a complete workflow for real-time qRT-PCR analysis directly from cultured cells without RNA purification. Featuring a unique method for lysing cultured cells while removing genomic DNA (gDNA) and preserving RNA integrity, the kits contain reverse transcription (RT) reagents for cDNA synthesis and either probe-based (TaqMan® probes) or dye-based (SYBR® Green dye) master mixes for real-time PCR analysis.
The 1-step and 2-step kits differ in that the 1-step kits combine reverse transcription and real-time PCR into a single step, significantly reducing the number of pipetting steps and protocol time.
Whether you are using plates or tubes, the Cells-to-CT™ Kits use a simple 7–8 minute sample preparation procedure outlined below (Figure 1). The lysis technology is designed for 10–100,000 cultured cells per sample. Cells are washed in PBS, then lysed for 5 minutes at room temperature; DNase treatment can be performed simultaneously. Lysis is terminated at room temperature by a 2-minute incubation with Stop Solution. The lysates are now ready for analysis on your real-time PCR instrument, or they can be stored at –20°C for up to 5 months. The 2-step kit workflow proceeds with reverse transcription to synthesize cDNA, after which the cDNA samples can be either archived or analyzed directly by real-time qPCR.
Because samples can be processed directly in culture wells (96- or 384-wells), sample handling and the potential for sample loss or transfer error are minimized, facilitating rapid high-throughput processing. Unlike traditional multi-step RNA isolation protocols, no heating, washing, or centrifugation steps are required. The kit greatly simplifies a laborious 30–60 minute process and reduces it to 7–8 minutes of reaction time, allowing complete cell culture processing from sample to qRT-PCR answer in as little as 35 minutes (e.g., Cells-to-CT™ 1-Step TaqMan® Kit).
Figure 1. The Cells-to-CT™ Kit procedure. The 1-step and 2-step kits differ in that the 1-step kits combine reverse transcription and real-time PCR into a single step, significantly reducing the number of pipetting steps and protocol time.
All components of the Cells-to-CT™ Kits have been optimized for consistent and reliable performance. This minimizes the guesswork involved in assembling separate sample preparation and real-time RT-PCR master mixes.
For added quality assurance, the Cells-to-CT™ 1-Step TaqMan® Kit has been validated with TaqMan® Gene Expression Assays, and the Cells-to-CT™ 1-Step Power SYBR® Green Kit has been validated with a set of primers for common gene targets. Both show performance equivalent to that obtained with purified RNA (Figure 2).
The performance of the TaqMan® Gene Expression Cells-to-CT™ Kit was compared to other commercially available lysis kits and to purified RNA. Inputs of 100–100,000 cells per lysis reaction were examined. The sensitivity of the TaqMan® Cells-to-CT™ Kit protocol was equivalent to that obtained with purified RNA, and it surpassed those from other suppliers (Figure 3).
|Figure 2. Sensitivity and detection of limited target sequences using Cells-to-CT™ Kits. (A) Sensitivity of gene expression assays with lysate vs. purified RNA. HeLa cells (10–100,000) were processed in triplicate using the Cells-to-CT™ 1-Step TaqMan® Kit, or RNA was purified using an RNA spin column method. Each set of samples was analyzed by real-time RT-PCR on the 7900HT Real-Time PCR System, for XENO and ACTB from the Cells-to-CT™ Control Kit. (B) Detection of limited target sequences. Increasing amounts of NIH3T3 cells (mouse) were added to 10,000 HeLa cells. The cells were processed in triplicate using the TaqMan® Gene Expression Cells-to-CT™ Kit (2-step procedure) or purified reagents with an RNA spin column method. All samples were reverse transcribed for mouse-specific (B2M) and human-specific (TKT) genes in triplicate reactions on a 7900HT Fast Real-Time PCR System.
||Figure 3. Good sensitivity obtained in TaqMan® Gene Expression Assays with lysates generated using the Cells-to-CT™ Kit. HeLa cells (100–100,000) were prepared using either traditional RNA purification, the TaqMan® Gene Expression Cells-to-CT™ Kit, or other lysate methods from suppliers Q and S. Reverse transcription reactions were made with the maximum recommended sample input for each kit, and real-time PCR was performed in triplicate using a PPIA TaqMan® Gene Expression Assay.
For Research Use Only. Not for use in diagnostic procedures.