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Invitrogen™ Cells-to-CT™ Bulk Fast Advanced RT Reagents
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Catalog No. A39110
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A39110 2500 reactions
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Catalog No. A39110 Supplier Invitrogen™ Supplier No. A39110

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

Description

Includes

Cells-to-Ct 20X Fast Advanced RT enzyme mix and 2X RT buffer

Invitrogen Fast Advanced Cells-to-CT Bulk RT Reagents are separately-available, large-quantity versions of the reagents used for reverse transcription (RT) in the TaqMan Fast Advanced Cells-to-CT Kit and the SYBR Green Fast Advanced Cells-to-CT Kit.

Invitrogen Fast Advanced Cells-to-CT Bulk RT Reagents are separately-available, large-quantity versions of the reagents used for reverse transcription (RT) in the TaqMan Fast Advanced Cells-to-CT Kit and the SYBR Green Fast Advanced Cells-to-CT Kit (20X RT enzyme mix and 20X RT buffer).

Specifications

Concentration 20X
Content And Storage • 6.8 mL 20X Fast Advanced RT Enzyme Mix
• 68 mL 2X Fast Advanced RT Buffer

Store at -5° to -30°C.
Detection Method Primer-probe
GC-Rich PCR Performance High
Reaction Speed Fast
High-throughput Compatibility High-throughput Compatible
Product Line Cells-to-CT
Product Type Bulk Fast Advanced Rt Reagent
Quantity 2500 reactions
Shipping Condition Dry Ice
Sufficient For 2500 Reactions
For Use With (Application) Gene Expression
Fidelity (vs. Taq) 20X
No. of Reactions 2500 Reactions
Purification Target Lysate
Sample Type Cell
Volume 68 mL
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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.
What is the size of the cDNA produced by the reverse transcriptase in the Cells-to-CT Bulk Fast Advanced RT Reagents?

The reverse transcriptase in this kit can make cDNA up to 7 Kb. However, this was not specifically tested on Cells-to-Ct lysates.
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
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.