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Invitrogen™ SYBR™ Green Fast Advanced Cells-to-CT™ Kit
Description
Includes
Cell lysis reagent, DNAse I, Stop Solution, 20X RT enzyme mix, 2X RT Buffer, Cells-to-Ct SYBR Fast Advanced Master Mix
The SYBR Green Fast Advanced Cells-to-Ct Kit is a complete cell lysate system designed for gene expression analysis directly from cultured cells without RNA purification. SYBR Green Fast Advanced Cells-to-Ct cell lysis reagents are supplied with highest sensitivity Cells-to-Ct Fast Advanced reverse transcription enzyme mix, 2X RT buffer, and best-in-class PowerUp SYBR Green Master mix. The SYBR Green Fast Advanced Cells-to-Ct Kit gives you the sensitivity you need in the fastest workflow available: get gene expressions analysis results from an entire plate of cells in about 1.5 hours.
- Complete—optimized workflow includes cell lysis reagents with gDNA removal, new Fast Advanced RT enzyme mix, buffer, and new PowerUp SYBR Green Fast Advanced Master Mix
- Fast—7-minute sample prep, including DNase treatment, at room temperature
- Easy—lyse samples in a tube or directly in culture plates
- Robust—perform gene expression analysis on 10–100,000 cells per sample; results equivalent to those from purified RNA
- Efficient—contains sufficient reagents to generate 1000 real-time PCR results from 100 starting samples
Unique method eliminates the need to isolate RNA from cultured cells
The Cells-to-Ct method features a unique lysis reagent that lyses cells and protects RNA. DNA removal occurs simultaneously in the 5-minute lysis step to save time and improve ease of use. Lysis is then stopped by the addition of a 'Stop Solution' that irreversibly binds to the lysis reagents.
The SYBR Green Fast Advanced Cells-to-CT Kit contains Cells-to-Ct Fast Advanced reverse transcription (RT) reagents for cDNA synthesis and PowerUp SYBR Green Master Mix for real-time PCR analysis. Primer sets are provided by the user.
Simple 7-minute sample preparation
The SYBR Green Fast Advanced Cells-to-CT Kit is designed for 10–100,000 cultured cells/sample. Cells are washed in PBS and lysed in lysis solution for five 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 reverse transcription or storage at –20°C for a short term or -80C for a long term.
Maximize sensitivity of detection
Unlike some competitor kits that limit the amount of lysate in the RT reaction to 10%, the SYBR Green Fast Advanced Cells-to-CT Kit can accommodate 45% of the total RT reaction volume as cell lysate. Additionally, cDNA can comprise up to 30% of the real-time PCR reaction volume. The large lysate volume in the optimized RT reaction, along with the large cDNA volume in the subsequent real-time PCR using the PowerUP SYBR Green Master Mix, lead to maximum sensitivity. The master mix amplifies the target precisely and accurately, enabling the detection of small quantities of target, such as transcripts expressed at low levels.
Reduced sample loss and transfer error
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 old-fashioned, 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 minutes.
Proven performance, proven together
All components of the SYBR Green Fast Advanced Cells-to-CT Kit have been optimized for consistent and reliable performance. This removes the guesswork involved in assembling separate sample preparation, RT, and real-time PCR kits, giving you the sensitivity you need in the fastest sample-to-answer workflow available.
Specifications
Specifications
| Concentration | 2X |
| Content And Storage | 1 mL 20X Fast Advanced RT Enzyme Mix, 20 mL 2X Fast Advanced RT Buffer, 20 mL Lysis Solution, 1 mL Stop Solution, 220 μL DNase I, 20 mL PowerUp SYBR Green Master Mix, Store at -5 to -30°C. |
| Detection Method | SYBR |
| Format | Tube |
| GC-Rich PCR Performance | High |
| PCR Method | 2-step RT-qPCR |
| Reaction Speed | 7 min. |
| DNase | 220 μL DNase I |
| High-throughput Compatibility | High-throughput Compatible |
| Product Line | Cells-to-CT |
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Frequently Asked Questions (FAQs)
There is no reason why the Cells-to-CT system shouldn’t work with any cell line. However, due to differences in cell size and composition, the maximum number of cells per lysis reaction may be slightly different for different cell lines. We recommend testing for inhibition and optimal cell input by using the TaqMan Cells-to-CT and SYBR Green Cells-to-CT Control kits.
Yes, the Stop Solution provided in the 2-step Cells-to-CT kits contains RNase inhibitor.
The TaqMan Gene Expression Cells-to-CT kit has been validated for duplexing. If you want to set up a multiplex real-time PCR reaction with 3 assays, we recommend using the TaqMan Fast Advanced Cells-to-CT kit (https://www.thermofisher.com/order/catalog/product/A35374).
To prevent signal from genomic DNA in the Cells-to-CT real-time PCR reaction, we recommend using a TaqMan assay or primer set that spans an exon-exon boundary, and adding DNase I to degrade genomic DNA during the lysis reaction. For optimal DNase activity in the lysis reaction, we recommend the following:
1. Ensure all media is removed from the cells.
2. Wash each well or cell pellet with an equal volume of room temperature 1X PBS.
3. Ensure the lysis reaction happens at room temperature. The lysis reaction may not reach room temperature if the plate is on ice prior to adding Lysis Solution, or cold Lysis Solution is added.
4. Warm the Lysis Solution to room temperature before adding to the cells.
5. Perform the lysis reaction at 25 degrees C for up to 8 minutes.
The Cells-to-CT Stop Solution prevents the DNase from being active, even if you add more. If you need to perform additional DNase treatment of the cell lysate sample after the Stop Solution is added, we recommend purifying the RNA from the cell lysate using traditional methods and DNase-treating the purified RNA.
For Research Use Only. Not for use in diagnostic procedures.