TaqMan™ Cells-to-CT™ Express Lysis Reagents - FAQs

Yes. Amplification of RNA targets directly from lysates using the TaqMan Cells-to-CT Express Kit consistently produces better results (lower Ct values) in side-by-side comparisons with column-purified RNA for low sample inputs. For very low sample inputs (fewer than 10 cells), we recommend using Single Cell-to-CT qRT-PCR Kit (Cat. No. 4458236, 4458237).

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The Express Lysis Solution included in the TaqMan Cells-to-CT Express Kit has been reformulated with components that do not require the use of a Stop Solution and yet provide the same level of performance as existing Cells-to-CT kits.

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This has not been tested. We recommend performing the Pilot Experiment explained in the product manual to determine overall compatibility, maximum input of cells without inhibition, and minimum input of cells for gene detection.

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Ensure that extra precautions are taken to prevent contamination. Confirm that the qPCR cycling includes the 2 mins at 50 degrees C step for UNG to remove contaminating amplicons. Spray down the area and pipets with DNAZap PCR DNA Degradation Solutions (Cat. No. AM9890) to remove contaminating DNA in your lab.

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Yes, to isolate DNA, we recommend omitting the Express ezDNase from the Express Lysis Solution. Note that for high cell inputs, the omission of Express ezDNase may cause the lysate to become viscous. We recommend performing a trial experiment before proceeding with actual samples.

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We have not evaluated the use of smaller lysis reaction volumes with the TaqMan Cells-to-CT Express Kit and recommend using the standard 50 µL lysis reaction volume.

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With the TaqMan Cells-to-CT Express Kit, up to 45% of the qPCR reaction volume can be cDNA.

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The TaqMan Cells-to-CT Express Kit displays equivalent sensitivity to other Cells-to-CT kits.

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Yes. Every component of the TaqMan Cells-to-CT Express Kit is REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals)-compliant and non-hazardous. Additionally, there is a significant reduction in plastic waste compared to using column-purified RNA.

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The TaqMan Cells-to-CT Express Kit has been evaluated using a wide diversity of gene assays and we have seen equivalent performance when compared to results obtained with traditionally purified RNA.

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The Express Lysis Solution can be stored at room temperature or 4 degrees C.

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Yes, the TaqMan Cells-to-CT Express Kit includes the SuperScript IV VILO Master Mix No-RT Control which can be used to determine if the sample contains genomic DNA. For a positive control, we recommend assaying for a highly expressed housekeeping gene as outlined in the pilot experiment in the product manual.

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The TaqMan Cells-to-CT Express Kit includes the SuperScript IV VILO Master Mix which contains oligo (DT)18 and random hexamer primers. This primer mix is able to reverse transcribe all cellular RNAs.

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Yes, the Express Lysis Solution contained in the TaqMan Cells-to-CT Express Kit has been formulated to effectively inhibit RNases.

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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.

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Yes, the TaqMan Cells-to-CT Express Kit is compatible with TaqMan Array plates and cards. We suggest using the recommended input of cDNA as outlined in the TaqMan Array plates and cards protocols.

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The TaqMan Cells-to-CT Express Kit contains TaqMan Fast Advanced Master Mix and should be compatible with the same instruments listed for the TaqMan Fast Advanced Master Mix, i.e., QuantStudio 3, 5, 6 Pro, 7 Pro, 6 Flex, 7 Flex and 12K Flex instruments. We have tested the TaqMan Cells-to-CT Express Kit with QuantStudio 5, 7 Flex, and 12K Flex instruments.

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With the TaqMan Cells-to-CT Express Kit, lysis reactions larger than the standard 50 µL volume have not been tested but should be compatible. Users should expect higher CT values due to increased sample dilution.

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The TaqMan Cells-to-CT Express Kit includes the SuperScript IV VILO Master Mix.

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The Express Lysis Solution contained in the TaqMan Cells-to-CT Express Kit has been reformulated to not require the use of a Stop Solution and to be REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals)-compliant and free of hazardous materials.

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The TaqMan Cells-to-CT Express Kit includes the TaqMan Fast Advanced Master Mix (Cat. No. 4444965) which is the same qPCR master mix included in the TaqMan Fast Advanced Cells-to-CT Kit (Cat. No. A35374, A35377, A35378).

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The two-step RT-qPCR workflow of the TaqMan Cells-to-CT Express Kit enables the user to reverse transcribe the entire transcriptome in one RT reaction and then perform separate qPCR reactions for any gene of interest. Two-step RT-qPCR provides the ability to archive cDNA, analyze multiple genes, and gives greater flexibility. Please see this table for an in-depth comparison.

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The Express Lysis Solution has been tested to withstand at least 5 freeze/thaw cycles. It can be stored at room temperature or 4 degrees C for up to a year if using often.

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Yes, the TaqMan Cells-to-CT Express Kit protocol allows for the following stopping points:

- After cell lysis: Cell lysates can be safely stored on ice for up to 5 hours or frozen at -80 degrees C for up to 1 year.
Additionally, high input cell lysates (~100,000 cells) are stable at room temperature for up to 30 mins while lower input cell lysates (<10,000 cells) are stable at room temperature for up to 1 hour.
- After assembly of the RT reaction: Assembled RT reactions can be stored on ice for up to 9 hours.
- After completion of the RT reaction: Completed RT reactions can be stored at -80 degrees C for up to 1 year.
- After assembly of the qPCR reaction: Assembled qPCR reactions can be stored at room temperature for up to 72 hours.

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Yes. When freezing cells, make sure to wash cells with PBS prior to freezing and remove as much PBS as possible leaving only the cell monolayer or pellet.

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The TaqMan Cells-to-CT Express Kit demonstrates equivalent performance, over a broad set of target genes and cell inputs, when compared to results obtained using traditionally purified RNA. Comparison data can be found on the product page. The TaqMan Cells-to-CT Express Kit tends to perform better than column-purified RNA at lower cell inputs because some amount of RNA is lost during the purification process.

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For most cell lines, the acceptable range of cell input is 10 - 100,000 cells. However, due to differences in cell size and composition, the maximum number of cells that can be used per lysis reaction may differ among cell types. We recommend performing the cell-input optimization pilot experiment as described in the product manual to determine the optimal range of cell input for your cells of interest.

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The TaqMan Fast Advanced Master Mix for qPCR included in the TaqMan Cells-to-CT Express Kit is compatible with multiplexed qPCR, however the TaqMan Cells-to-CT Express Kit has not been evaluated for multiplexing. We expect that it should work but users would need to evaluate with their own assays.

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Yes. The Express Lysis Solution and Express ezDNase can be purchased in bulk as TaqMan Cells-to-CT Express Bulk Lysis Reagents (Cat. No. A57989). The standalone ezDNase Enzyme (Cat. No. 11766051), SuperScript IV VILO Master Mix (Cat. No. 11756050), and TaqMan Fast Advanced Master Mix for qPCR (Cat. No. 4444556) can also be purchased separately.

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Yes, cDNA generated with the TaqMan Cells-to-CT Express Kit is stable at -80 degrees C for at least 1 year. We have also shown that cDNA can go through five freeze/thaw cycles without any loss in signal.

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Yes, lysates from the TaqMan Cells-to-CT Express Kit are stable at -80 degrees C for at least 1 year. We have also shown that lysates can go through five freeze/thaw cycles without any loss in signal.

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gDNA contamination can be identified by performing an RT control reaction without the RT enzyme. The TaqMan Cells-to-CT Express Kit contains the SuperScript IV VILO Master Mix No-RT Control which includes all components except for the RT enzyme and can be used for monitoring of gDNA contamination. To avoid the detection of gDNA, we recommend using a TaqMan Gene Expression Assay specifically designed to span an intron-exon boundary. Such assays are designated with an "_m1" suffix. More information can be found here.

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Yes, the TaqMan Cells-to-CT Express Kit includes Express ezDNase which can be added to the Express Lysis Solution for removal of gDNA during cell lysis. Express ezDNase is a double-strand specific, heat-labile DNase that will only digest gDNA, making it compatible with the downstream reverse transcription reaction. The enzyme is automatically inactivated during a heat kill step included in the reverse transcription program. To avoid the detection of gDNA, we recommend using a TaqMan Gene Expression Assay specifically designed to span an intron-exon boundary. Such assays are designated with an "_m1" suffix. More information can be found here.

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The TaqMan Cells-to-CT Express Kit has been shown to be compatible with a wide variety of cell lines. A continuously updated list of cell lines that have been tested and shown to be compatible can be found on the product page. Note that due to differences in cell size and composition, the maximum number of cells that can be used per lysis reaction may differ between cell lines. We recommend performing the pilot experiment described in the product manual to determine if your cell line of interest is compatible with the kit.

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The TaqMan Cells-to-CT Express Kit has been tested using 96-well and 384-well plate formats using the standard 50 µL lysis reaction. If larger well sizes are desired, we recommend scaling up the volume of lysis reaction accordingly and performing a validation experiment.

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Yes, the TaqMan Cells-to-CT Express Kit can be used with both adherent and suspensions cells. Protocols are provided for both adherent and suspension cells.

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Yes, the lysate preparation step in the TaqMan Cells-to-CT Express Kit protocol is completed at room temperature and does not require any centrifugation steps, making it very automation friendly.

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Yes, the TaqMan Cells-to-CT Express Kit allows for direct RT-qPCR amplification from cell lysates without purification of RNA.

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