Complete—Kits include cell lysis reagents with gDNA removal, RT enzyme mix, buffer, and Power SYBR® Green or Fast SYBR Green Master Mix
Easy—Lyse samples in a tube or directly in culture plates
Fast—7-minute sample preparation, including DNase treatment, all at room temperature
Robust—Perform gene expression analysis on 10–100,000 cells per sample with results equivalent to those from purified RNA
Originally designed for the TaqMan® Gene Expression system and now available for SYBR Green-based real-time PCR, the new Power SYBR Green and Fast SYBR Green Cells-to-CT Kits take you from cells in culture to real-time RT-PCR analysis without RNA purification. The kits use a unique method for lysing cultured cells while simultaneously removing genomic DNA and preserving RNA integrity. These simple, complete workflows are economical alternatives to using separate sample preparation, RT, and real-time PCR kits. They provide robust performance because all components are optimized to work together, and have been extensively tested for sensitivity and specificity with a broad selection of primers. The workflow is suitable for a few samples or automated, high throughput applications, and saves time over traditional methods (Figure 1).
Figure 1. Time Comparison of SYBR® Green-based Cells-to-CT™ Kits with Traditional Workflows Requiring RNA Purification.
Simple 7-Minute Sample Preparation: Part of a Complete Workflow
The SYBR Green Cells-to-CT Kits use a simple 7-minute sample preparation procedure. Starting with 10–100,000 cultured cells per sample, cells are washed in PBS, and then lysed for 5 minutes at room temperature; DNase treatment can be performed simultaneously. Lysis is terminated at room temperature by adding Stop Solution and incubating for 2 minutes. The lysates are now ready for reverse transcription or storage at –20°C for up to 5 months. Because samples can be processed directly in culture plates (96- or 384-well), sample handling and the potential for sample loss or transfer error are minimized, facilitating rapid, high throughput processing. Unlike old-fashioned multi-step RNA isolation protocols, no heating, washing, or centrifugation steps are required. The kit simplifies a laborious 30–60 minute process and reduces it to 7 minutes. After the sample lysate is prepared, a portion of it is used in a reverse transcription (RT) reaction, followed by a real-time PCR using either Power SYBR Green or Fast SYBR Green Master Mixes.
The SYBR Green Cells-to-CT Kits demonstrate performance equivalent to that of purified RNA, as shown by by high correlation of real-time PCR data obtained from both methods (Figure 3). All the required reagents are included in the kits, with the exception of PCR primer sets specific for your targets of interest.
Figure 3. Power SYBR® Green and Fast SYBR® Green Cells-to-CT™ Kits Perform Equivalently to Purified RNA Over a Broad Set of Gene Targets.
A cell mixture (10,000 cells) comprised of HeLa, HegG2, Jurkat, HEK-293, and U-87-MG cells were analyzed using either traditional RNA purification and real-time PCR, or the Power SYBR Green and Fast SYBR Green Cells-to-CT Kits. Technical (real-time PCR) quadruplicates were performed for each method for each of the 155 primer sets. A high correlation was observed between the traditional purified RNA real-time PCR results and results obtained using the Power SYBR Green Cells-to-CT Kit (y = 0.94x + 1.46, R2= 0.96) and Fast SYBR Green Cells-to-CT Kit (y = 0.97x +0.45, R2=0.96).
Achieve Superior Sensitivity and Dynamic Range
The Power SYBR Green Cells-to-CT Kit includes Power SYBR Green PCR Master Mix for highly sensitive nucleic acid quantitation, enabling the detection of as few as 2 copies of a target gene over a broad range of input amounts. The formulation contains highly purified AmpliTaq Gold® DNA Polymerase, LD for increased sensitivity compared to other polymerases. The Power SYBR Green Cells-to-CT Kit delivers superior sensitivity and reproducibility without compromising specificity or dynamic range. The sensitivity of the Power SYBR Green Cells-to-CT method was equivalent to that obtained with purified RNA (Figure 4), and surpassed competitor lysates. Furthermore, the lack of inhibition at high cellular inputs and the low variation in technical replicates demonstrate the high reproducibility and robustness of the kit, due in part to the superior performance characteristics of the Power SYBR Green Master Mix.
Figure 4. Sensitivity of the Power and Fast SYBR® Green Cells-to-CT™ Kits Versus Competing Technologies.
HeLa cells (100–100,000) were prepared using either traditional RNA isolation, the Power or Fast SYBR® Green Cells-to-CT™ Kit protocols, or other lysate methods from Competitors I and Q. Real-time PCR was performed in triplicate using a primer set for PPIA.
Make it Even Faster with Fast SYBR Green Master Mix
The Fast SYBR Green Cells-to-CT Kit has all the advantages of the Power SYBR Green Cells-to-CT Kit with the additional benefit provided by AmpliTaq® Fast DNA Polymerase, UP. This polymerase is designed for instant hot start, which minimizes nonspecific product formation and allows reactions to be set up at room temperature. The Fast SYBR Green Cells-to-CT Kit is designed to deliver PCR results on fast-enabled real-time instruments in less than half the time of standard real-time PCR reagents and cycling conditions, while maintaining equivalent performance. Figure 4 illustrates the dynamic range and sensitivity of Fast SYBR Green Cells-to-CT Kit, comparable to Power SYBR Green PCR Cells-to-CT Kit. The Fast SYBR Green Cells-to-CT Kit workflow reduces the time from sample to result by more than 50%, compared to traditional RNA purification and standard real-time PCR (Figure 1).
Whichever kit you choose, the new Power SYBR Green and Fast SYBR Green Cells-to-CT Kits provide quick, easy, and complete solutions for real-time RT-PCR from cells.
Nathan Harris, Annmarie Schramm, Richard Fekete, Annie Nguyen • Applied Biosystems Inc., Austin, TX