TaqMan™ Fast Advanced Cells-to-CT™ Kit, 400 reactions - FAQs

View additional product information for TaqMan™ Fast Advanced Cells-to-CT™ Kit - FAQs (A35377, A35378, A35374)

22 product FAQs found

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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 (Cat. No. 4444557) can be used in place of the TaqMan Gene Expression Master Mix (Cat. No. 4369016) 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.

Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.

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