Ambion Cells-to-CT Kits
Invitrogen Ambion Cells-to-CT™ Kits allow you to measure relative gene expression by real-time RT-PCR without having to purify RNA prior to amplification. Cells-to-CT Kits are available for both Invitrogen™ TaqMan® and Invitrogen SYBR Green detection.
1-step or 2 step qRT-PCR?
Use 1-step qRT-PCR kits when you need speed and convenience, e.g., high-throughput drug screening or siRNA validation. Use 2-step qRT-PCR kits for optimal flexibility, e.g., for cDNA archiving or ability to choose your amplification system.
TaqMan or SYBR Green chemistry?
TaqMan detection typically offers a shorter protocol, higher specificity and sensitivity, and the ability to multiplex. SYBR Green dye–based methods are often regarded as more flexible in terms of the range of primers that can be used, and the cost is typically lower.
Studying single-cell gene expression?
Our Single Cell-to-CT Kit is a complete kit with an easy-to-follow protocol designed for maximum sensitivity when studying single-cell gene expression. More on the Single Cell-to-CT Kit
|Order Now||Order Now||Order Now||Order Now||Order Now|
|Cells-to-CT 1-Step TaqMan Kit||Cells-to-CT 1-Step Power SYBR Green Kit||TaqMan Gene Expression Cells-to-CT Kit||Power SYBR Green Cells-to-CT Kit||Single Cell-to-CT qRT-PCR Kit|
|1-step qPCR sample prep||1-step qPCR sample prep||2-step qPCR sample prep||2-step qPCR sample prep||Single Cell-to-CT™ Kit|
|Detection chemistry||TaqMan||SYBR Green||TaqMan||SYBR Green||TaqMan|
|No. of cells in lysis step||10–100,000 cells||10–100,000 cells||10–100,000 cells||10–100,000 cells||1–10 cells|
|Detection sensitivity||1–10 copies||Variable*||1–10 copies||Variable*||1–10 copies|
|RNA purification required||No||No||No||No||No|
|Sample prep time (to qRT-PCR) for 8 samples||7 min||7 min||7 min||7 min||7 min|
|Total time (from cell lysis to qRT-PCR results)||35 min||1 hr 23 min||2 hr 15 min||2 hr 45 min||3 hr|
|Includes RT enzyme and master mix||✓||✓||✓||✓||✓|
|Includes preamplification reagents||✓|
|Suitable for automation||✓||✓||✓||✓||✓|
* Depends on template quality and primer/design optimization.
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.|
|The TaqMan Gene Expression Cells-to-CT Kit has performed consistently. The performance is increased as compared to RNA purification, RT steps, and then qRT-PCR. There is less handling, less chance at mistakes. Because of this time savings and increase in sensitivity, I’ve incorporated this protocol into the Human Embryonic Stem Cell Training Course at the Stem Cell Research Center at Rutgers University.|
—Dr. Rick Cohen, Director, hESC Core Facility, Stem Cell Research Center, Rutgers University, Piscataway, NJ
|Gene expression quantification on cultured cells using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) typically involves an RNA purification step that limits sample processing throughput and precludes parallel analysis of large numbers of samples. An approach in which cDNA synthesis is carried out on crude cell lysates instead of on purified RNA samples can offer a fast and straightforward alternative. Here, we evaluate such an approach, benchmarking the Ambion Cells-to-CT kit with the classic workflow of RNA purification and cDNA synthesis, and demonstrate its good accuracy and superior sensitivity.|