Single cell analysis has the potential to transform life science research. The standard practice of averaging gene expression results from many cells can mask subtle, important changes occurring within single cells. To further understand the complexity of biological systems, single cell analysis must be performed. The Ambion® Single Cell-to-C_T™ Kit contains a complete validated workflow for gene expression analysis for samples containing between 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 5 steps.

Key product features:

• Pre-optimized workflow for Real-Time RT-PCR from single cells
• Maximum sensitivity for single cell analysis
• Superior performance and reproducibility than alternatives
• Complete kit convenience and easy to follow protocol

Pre-optimized Protocol Ensures Success and Saves Time!
The Ambion® Single Cell-to-C_T™ Kit presents a complete validated workflow for gene expression analysis for samples containing between 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 5 steps (Figure 1). 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 VILO™ with Superscript 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 Accomplished
Sensitivity of real-time PCR results can be affected by the efficiency of sample preparation, reverse transcription or amplification. The Ambion® Single Cell-to-C_T™ 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-C_T™ Kit demonstrates appropriate linearity and sensitivity of qPCR results compared to an 100 cell sample control with excellent technical reproducibility (Figure 2). 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. Figure 3 demonstrates the accuracy and signal enhancement of a 96-gene panel using pre-amplification.

Superior Performance and Reproducibility Over Alternatives
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 carry over, including gDNA contamination, which can affect all qRT-PCR applications. In contrast, the Ambion® Single Cell-to-C_T™ Kit shows superior sensitivity and reproducibility for single cell analysis compared to traditional purification or homebrew boiling methods (Figure 4).