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  7. Invitrogen™ TaqMan™ Fast Advanced Cells-to-CT™ Kit

Invitrogen™ TaqMan™ Fast Advanced Cells-to-CT™ Kit
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Catalog No. A35378
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A35378 400 Reactions
A35374 40 Reactions
A35377 100 Reactions
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Catalog No. A35378 Supplier Invitrogen™ Supplier No. A35378

Please call Customer Service at 1-800-234-7437 or send an email to help@thermofisher.com for assistance.

Description

Includes

Cell lysis reagent, DNAse I, Stop Solution, 20X RT enzyme mix, 2X RT Buffer, TaqMan Fast Advanced Master Mix

The TaqMan Fast Advanced Cells-to-Ct Kit is a complete cell lysate system designed for gene expression analysis directly from cultured cells without RNA purification.

The TaqMan Fast Advanced Cells-to-Ct Kit is a complete cell lysate system designed for gene expression analysis directly from cultured cells without RNA purification. TaqMan Fast Advanced Cells-to-Ct cell lysis reagents are supplied with highest sensitivity Fast Advanced reverse transcription enzyme mix, 2X RT buffer, and best-in-class TaqMan Fast Advanced master mix. The TaqMan Fast Advanced Cells-to-Ct Kit gives you all the sensitivity you need in the fastest workflow available: get gene expressions analysis results from an entire plate of cells in about 80 minutes.

The TaqMan Fast Advanced Cells-to-Ct Kit is a complete cell lysate system designed for gene expression analysis directly from cultured cells without RNA purification. TaqMan Fast Advanced Cells-to-Ct cell lysis reagents are supplied with highest sensitivity Fast Advanced reverse transcription enzyme mix, 2x RT buffer, and best-in-class TaqMan Fast Advanced master mix. The TaqMan Fast Advanced Cells-to-Ct Kit gives you all the sensitivity you need in the fastest workflow available: get gene expressions analysis results from an entire plate of cells in about 80 minutes.

  • Complete—optimized workflow includes cell lysis reagents with gDNA removal, new Fast Advanced RT enzyme mix, buffer, and new TaqMan 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 500 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 TaqMan Fast Advanced Cells-to-CT Kit contains reverse transcription (RT) reagents for cDNA synthesis and TaqMan Fast Advanced Master Mix for real-time PCR analysis. TaqMan primer/probe sets are sold separately.

Simple 7-minute sample preparation

The TaqMan Fast Advanced Cells-to-CT Kit is designed for 10–100,000 cultured cells/sample. Cells are washed in PBS and lysed in 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 -80°C 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 TaqMan Fast Advanced Cells-to-CT Kit can accommodate 45% of the total RT reaction volume as cell lysate. Additionally, cDNA can comprise up to 45% 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 TaqMan Fast Advanced 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.

Compatible with gold standard TaqMan Gene Expression Assays

The TaqMan Fast Advanced Cells-to-CT Kit workflow enables unsurpassed gene expression evaluation with any of the >700,000 TaqMan Gene Expression Assays. This kit has been extensively tested for specificity with a broad selection of TaqMan Gene Expression Assays.

Proven performance, proven together

All components of the TaqMan 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. And the TaqMan Fast Advanced Cells-to-CT Kit has been validated with TaqMan Gene Expression Assays for added quality assurance, giving you the sensitivity you need in the fastest sample-to-answer workflow available.

Specifications

Concentration 2X
Content And Storage 20X Fast Advanced RT Enzyme Mix, 1 mL, 2X Fast Advanced RT Buffer, 20 mL, Lysis Solution, 20 mL, Stop Solution, 2 x 1 mL, DNase I, 220 μL, TaqMan Fast Advanced Master Mix, 20 mL, Store at -5°C to -30°C.
Detection Method TaqMan
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
Product Type Fast Advanced Cells-to-Ct Kit
Quantity 400 reactions
Shipping Condition Dry Ice
Sufficient For 400 Reactions
For Use With (Application) Gene Expression
Fidelity (vs. Taq) 2X
No. of Reactions 400 Reactions
Purification Target Lysate
Reagent Type Cell Lysis
Sample Type Mammalian cells
Starting Material 10-100,000 Cells
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Frequently Asked Questions (FAQs)

Will the Cells-to-CT system work with my special cell line?

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.
I purchased the TaqMan Fast Advanced Cells-to-CT Kit and am seeing precipitates in the TaqMan Fast Advanced Master Mix. Should I be concerned?

The TaqMan Fast Advanced Master Mix can develop particulates that float in solution. Such particulates do not affect the kit performance.
Which reverse transcription primers are used in the Fast Advanced Cells-to-CT Kits (Cat. No. A35374, A35379)?

Oligo dT and random primer (octamer) mix.
Can I use the Cells-to-CT Bulk Lysis Reagents (Cat. No. 4391851C) with the Fast Advanced Cells-to-CT Kits (Cat. No. A35374, A35379)?

Yes.
Are the TaqMan Cells-to-CT Control Kit (Cat. No. 4386995) and the SYBR Green Cells-to-CT Control Kit (Cat. No. 4402959) compatible with the Fast Advanced Cells-to-CT products?

Yes. The Cells-to-CT control kits contain Xeno RNA and assays/primers for detecting Xeno RNA and B-actin. They can be used for troubleshooting and setting up a pilot experiment to determine optimal cell input.
Can the Fast Advanced Cells-to-CT Kit (Cat. No. A35374, A35379) lysates be used with the legacy Cells-to-CT kits, and vice versa?

Yes. The legacy Cells-to-CT kits and the Fast Advanced Cells-to-CT kits use the same lysis solution, stop solution, and DNase I.
Can I use the Express Lysis Solution included in the TaqMan Cells-to-CT Express Kit with the TaqMan Fast Advanced Cells-to-CT kits?

The TaqMan Cells-to-CT Express kit has been optimized to work well as a full workflow. We do not recommend using the Express Lysis Solution contained in the TaqMan Cells-to-CT Express Kit with any other Cells-to-CT kit.

Do the 2-step Cells-to-CT kits contain RNase inhibitor?

Yes, the Stop Solution provided in the 2-step Cells-to-CT kits contains RNase inhibitor.
Can I use the TaqMan Gene Expression Cells-to-CT kit for multiplexing?

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).

I am using a Cells-to-CT kit and I see signal from the genomic DNA in my real-time PCR results. How do I get rid of the genomic DNA contamination?

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.
Can I DNase-treat my Cells-to-CT cell lysate after the Stop Solution has been added to the lysis reaction?

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.
I am using the TaqMan Gene Expression Cells-to-CT kit. Can I use the TaqMan Fast Advanced Master Mix in place of the TaqMan Gene Expression Master Mix to set up the qPCR reaction?

Yes, the TaqMan Fast Advanced Master Mix can be used in place of the TaqMan Gene Expression Master Mix when setting up the qPCR reaction for the TaqMan Gene Expression Cells-to-CT kit.
Can I use the Cells-to-CT kits with plasma samples?

We have not tested the compatibility of Cells-to-CT kits with plasma samples and would not recommend using this kit for plasma samples. Please refer to the following table to select an RNA purification kit based on your sample type: https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-types/total-rna-extraction.html.
What primers are used in the reverse transcription step for the Cells-to-CT kits?

The following 2-step Cells-to-CT kits use a mixture of oligo dT and random primers for the reverse transcription step:
• TaqMan Fast Advanced Cells-to-CT Kit
• TaqMan Gene Expression Cells-to-CT Kit
• SYBR Green Fast Advanced Cells-to-CT Kit
• Power SYBR Green Cells-to-CT Kit
• Fast SYBR Green Cells-to-CT Kit

The following 1-step Cells-to CT kits require gene-specific primers for the reverse transcription step, so you can either use your SYBR qPCR primers or TaqMan assay primers:
• Cells-to-CT 1-Step TaqMan Kit
• Cells-to-CT 1-Step Power SYBR Green Kit
Can I scale down the lysis reaction volume for the Cells-to-CT kits?

We do not recommend scaling down the lysis reaction volume for the Cells-to-CT kits. Reducing the lysis reaction volume below 50 µL can lead to incomplete inactivation of reagents and cause variability with results.
How do I remove gDNA from my Cells-to-CT reaction?

1. Ensure that all media is removed from the wells.
2. Wash with an equal volume of room temperature 1X PBS.
3. Ensure that the lysis reaction happens at room temperature (the lysis reaction may not reach room temperature if the plate is on ice, quickly moved to the bench, or cold lysis solution is added).
4. Warm the lysis solution to room temperature before adding to the cells.
5. Perform lysis reaction at 25 degrees C for up to 8 mins.
Can the Cells-to-CT cell lysis reagents be used with the Fast Advanced Cells-to-CT kits?

Yes. The Fast Advanced Cells-to-CT kits use the same lysis solution, stop solution, and DNase I as the Cells-to-CT kits.
Can the Fast Advanced Cells-to-CT kits be used for multiplexing?

The TaqMan Fast Advanced Cells-to-CT Kit is compatible with multiplex gene expression experiments. The SYBR Green Fast Advanced Cells-to-CT Kit does not have multiplexing capability.
Are the TaqMan Cells-to-CT Control Kit and the SYBR Green Cells-to-CT Control Kit compatible with the Fast Advanced Cells-to-CT kits?

Yes. The TaqMan Cells-to-CT Control Kit and SYBR Green Cells-to-CT Control Kit are compatible with the Fast Advanced Cells-to-CT kits and workflow.
Is the TaqMan Fast Advanced Cells-to-CT enzyme and buffer sold separately?

The RT enzyme and RT buffer provided in the Fast Advanced Cells-to-CT kits can be purchased in bulk (Cat. No. A39110, https://www.thermofisher.com/order/catalog/product/A39110).

What is the optimal number of cells to use per reaction with Cells-to-CT kits?

To identify the maximum number of cells to use for each reaction, we recommend testing a range of cellular input amounts by setting up a serial dilution. Instructions for determining the best cell number input with Fast Advanced Cells-to-CT kits can be found in the User Bulletin: Cell Input Optimization for SYBR Green Fast Advanced Cells-to-CT Kit (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017931_CellInputOptimization_SYBRGreenFastAdvCells-to-CT_UB.pdf) and the User Bulletin: Cell input optimization for TaqMan Fast Advanced Cells-to-CT Kit (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017932_CellInputOptimization_TaqManFastAdvCells-to-CT_UB.pdf). For the non-Fast Advanced Cells-to-CT kits, instructions can be found in the Pilot Experiment section of the protocol for each kit.
Which cell lines have been tested to work with the Cells-to-CT system?

Here is a short list of cell lines that have been tested with the Cells-to-CT system: HeLa, HepG2, primary hepatocytes, SK-N-AS, SK-N-SH, U-87 MG, ME-180, A549, Jurkat, PC-12, PT-K75, NIH/3T3, Raji, HEK-293, COS-7, CHO-K1, NCI-H460, DU-145, K562, U-2 OS, Huh-7, Neuro 2A, and BJ. For additional information please visit the following page: https://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/rna-extraction/rna-types/total-rna-extraction/cells-to-ct-kits/cells-to-ct-faq.html

For Research Use Only. Not for use in diagnostic procedures.