The Ambion Single Cell-to-CT™ Kit contains a complete validated workflow for gene expression analysis for samples containing 1–10 cells. Each kit contains reagents for sample preparation, reverse transcription, pre-amplification, and qPCR that have been optimized together in a simple workflow that can be completed in only five steps.
Pre-optimized protocol ensures success and saves time
Each workflow begins with a simple 7-minute sample prep, where cells are effectively and reproducibly lysed with minimal processing into a solution that is compatible with downstream RT-PCR, without the need for purification. This is followed by the use of Superscript™ RT for reverse transcription, the TaqMan™ PreAmp Master Mix to amplify the cDNA, and TaqMan Gene Expression Master Mix for qPCR. All four of these steps have been developed to provide maximum sensitivity. The entire cell sample is maintained in the same well throughout the procedure so there is no sample loss or dilution of the precious limited material that could affect sensitivity.
Single cell sensitivity
Sensitivity of real-time PCR results can be affected by the efficiency of sample preparation, reverse transcription, or amplification. The Single Cell-to-CT Kit has addressed each of these potential problem areas to create a solution that enables reliable and robust gene expression analysis from single cells with maximal sensitivity. Detection of single cell equivalents using the Single Cell-to-CT Kit demonstrates appropriate linearity and sensitivity of qPCR results compared to a 100 cell sample control with excellent technical reproducibility. As expected, individual single cells exhibit greater variability due to inherent biological heterogeneity. In addition, through inclusion of a cDNA pre-amplification step, targets of interest are accurately amplified prior to real-time PCR. This critical step boosts signal in an unbiased manner, extending the analysis potential of limited samples. The accuracy and signal enhancement of a 96-gene panel using pre-amplification is shown.
Superior performance and reproducibility compared to alternative methods
Traditional sample preparation methods using organic solvents, glass fiber filters, or magnetic beads are not suitable for single-cell analysis due to the loss of sample through incomplete RNA precipitation, binding, and elution. Additionally, most single-cell homebrew methods which involve simple boiling to lyse cells lack the ability to inactivate endogenous RNases to stabilize gene expression profiles and can result in chemical cleavage of the RNA and inhibitor carryover, including gDNA contamination, which can affect all qRT-PCR applications. In contrast, the Single Cell-to-CT Kit shows superior sensitivity and reproducibility for single-cell analysis compared to traditional purification or homebrew boiling methods.
Pre-optimized workflow for real-time RT-PCR from single cells
Maximum sensitivity for single-cell analysis
Superior performance and reproducibility compared to alternative methods
Complete kit convenience and easy-to-follow protocol
50 μL Single Cell DNase I, 0.5 mL Single Cell Lysis Solution (store at 4°C), 50 μL Single Cell Stop Solution, 75 μL Single Cell SuperScript™ RT, 5 mL TaqMan™ Gene Expression MasterMix (store at 4°C), 265 μL Single Cell Pre-Amp Mix, 150 μL Single Cell VILO™ RT Mix, Store kit components at -20°C except for Single Cell Stop Solution and TaqMan™ Gene Expression MasterMix, which should be stored at 4°C.
2-step RT-qPCR
7000 System, 7300 System, 7500 Fast System, 7900HT Fast System, 7900HT System, GeneAmp 9700, GeneAmp™ 2400, StepOne™, Fast Mode, StepOne™, Standard Mode, StepOnePlus™, Fast Mode, StepOnePlus™, Standard Mode, Veriti Thermal Cycler
Ambion™, Cells-to-CT™
50 reactions
RT-PCR
Mammalian Cells
TaqMan
7 min.
Fewer resources used and less waste, less hazardous
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