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  7. Invitrogen™ TaqMan™ Cells-to-CT™ Express Kit

Invitrogen™ TaqMan™ Cells-to-CT™ Express Kit
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Catalog No. A57988
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A57988 2500 Reactions
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Catalog No. A57988 Supplier Invitrogen™ Supplier No. A57988

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

Description

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

The TaqMan Cells-to-CT Express Kit is a complete cell lysate system designed for gene expression analysis directly from cultured cells without RNA purification. It enables RT-qPCR of the highest sensitivity and quickest workflow using just five components: Express Lysis Buffer, Express ezDNase, Express RT Master Mix, RT controls, and our best-in-class TaqMan Fast Advanced Master Mix. The TaqMan Cells-to-CT Express Kit provides the sensitivity you need in the fastest workflow available: RT-qPCR results for gene expression analysis in approximately 70 minutes.

Features of the TaqMan Cells-to-CT Express Kit include:

  • Extraordinary ease and speed—96 samples ready for RT-qPCR in as little as 5 minutes
  • No tedious RNA purification—no columns, heating, or centrifugation
  • Verified workflows—complete set of reagents optimized to work efficiently right out of the box
  • Environmentally friendly—the kit is REACH Compliant, has fewer components, and requires fewer pipetting steps reducing plastics costs
  • Right sized kits for automation—available in 2500 reaction size that includes both lysis and RT components

Optimized chemistry with no need for stop solution

Gene expression analysis from direct cell lysis is made simple with our TaqMan Cells-to-CT Express Kit. With only one step for cell lysis and no need for the addition of a Stop Solution, our Express technology enables a rapid workflow with sensitive, accurate, and reproducible results. 10–100,000 cells are lysed using the Express Lysis Solution. The optional inclusion of ezDNase to the Express Lysis Solution enables removal of genomic DNA during cell lysis. Following a 5-minute incubation at room temperature, the lysate is used directly for reverse transcription using SuperScript IV VILO Master Mix. Once the RT reaction is complete, qPCR analysis is carried out using TaqMan Fast Advanced Master Mix and a TaqMan Gene Expression Assay.

Streamlined workflow allowing for rapid, accurate and sensitive results

RNA prepared using the TaqMan Cells-to-CT Express technology substantially expedites and streamlines gene expression analysis. Cell lysates exhibit sensitivity and specificity better than or equivalent to RNA extracted using traditional spin column isolation methods. The lysis procedure simultaneously prepares cell lysates for RT-qPCR and removes genomic DNA in only five minutes. The lysis step is simple to automate with robotic platforms for high-throughput processing of 96- or 384-well plates because it takes place entirely at room temperature. The procedure is also economical as there are only a few pipetting steps and, for cells grown in 96- or 384-well plates, no sample transfers. The high efficiency SuperScript IV VILO RT Master Mix transcribes even the most difficult (degraded and low abundant) RNA while reducing the typical reaction time by 10 minutes. Additionally, the TaqMan Cells-to-CT Express Kit is compatible with over 700,000 TaqMan Gene Expression Assays.

Maximize sensitivity of detection

Unlike some competitor kits that limit the amount of lysate in the RT reaction to 10%, the TaqMan Cells-to-CT Express 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.

Specifications

Content And Storage Express Lysis Solution (room temperature or 4°C), Express ezDNase (-20°C), SuperScript IV VILO Master Mix (-20°C), SuperScript IV VILO No RT Control (-20°C), TaqMan Fast Advanced Master Mix (4°C)
Detection Method TaqMan
Format Kit
PCR Method 2-step RT-qPCR
High-throughput Compatibility Yes
Kit Type Direct Cell Lysis RT-qPCR
Quantity 2500 reactions
Shipping Condition Frozen, Dry Ice
Target cDNA
For Use With (Application) Gene Expression
Isolation Technology Direct cell lysis
No. of Reactions 2500 Reactions
Purification Target RNA
Sample Type Mammalian Cells
Starting Material 10-100,000 Cells
Includes Bulk kit includes Lysis and RT-qPCR reagents
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Frequently Asked Questions (FAQs)

I purchased the TaqMan Cells-to-CT Express 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.
Would the direct lysis approach to DNA/RNA analysis be a good alternative for very small samples if using the TaqMan Cells-to-CT Express Kit?

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).

Why don't I need a Stop Solution in the TaqMan Cells-to-CT Express Kit?

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.

Can I use the TaqMan Cells-to-CT Express Kit on uncultured primary cells?

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.

I'm seeing PCR products in the no-template PCR control when using the TaqMan Cells-to-CT Express Kit. How can I control contamination?

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.

Can I use the TaqMan Cells-to-CT Express Kit to isolate DNA and RNA?

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.
Can I scale down the volume of the lysis reaction with the TaqMan Cells-to-CT Express Kit?

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.

What is the maximum amount of cDNA that I can use in the qPCR reaction using the TaqMan Cells-to-CT Express Kit?

With the TaqMan Cells-to-CT Express Kit, up to 45% of the qPCR reaction volume can be cDNA.
How does the sensitivity of the TaqMan Cells-to-CT Express Kit compare to other Cells-to-CT kits?

The TaqMan Cells-to-CT Express Kit displays equivalent sensitivity to other Cells-to-CT kits.
Is the TaqMan Cells-to-CT Express Kit eco-friendly?

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.
Will the TaqMan Cells-to-CT Express Kit work for my gene target of interest?

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.
Can I store the Express Lysis Solution included in the TaqMan Cells-to-CT Express Kit at room temperature?

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

Does the TaqMan Cells-to-CT Express Kit include a control for RT reactions?

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.
What primers are used for reverse transcription in the TaqMan Cells-to-CT Express Kit?

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.
Does the TaqMan Cells-to-CT Express Kit contain RNase inhibitor?

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

Can I use the TaqMan Cells-to-CT Express Kit with TaqMan Arrays?

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.
Which real-time PCR instruments are compatible with the TaqMan Cells-to-CT Express Kit?

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.
Can I scale up the volume of the lysis reaction with the TaqMan Cells-to-CT Express Kit?

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.
Which reverse transcriptase is included in the TaqMan Cells-to-CT Express Kit?

The TaqMan Cells-to-CT Express Kit includes the SuperScript IV VILO Master Mix.
How does the Express Lysis Solution contained in the TaqMan Cells-to-CT Express Kit differ from lysis buffers from other Cells-to-CT kits?

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.
What is the qPCR master mix provided in the TaqMan Cells-to-CT Express Kit?

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).
What are the advantages of the two-step RT-qPCR workflow of the TaqMan Cells-to-CT Express Kit?

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.

How many times can I freeze and rethaw the Express Lysis Solution included in the TaqMan Cells-to-CT Express Kit?

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.

Does the TaqMan Cells-to-CT Express Kit protocol have any stopping points?

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.
Can I use frozen cells with the TaqMan Cells-to-CT Express Kit?

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.
How does the TaqMan Cells-to-CT Express Kit perform compared to traditional RNA purification methods?

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.
With the TaqMan Cells-to-CT Express Kit, what is the optimal number of cells to use in the lysis reaction?

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.
Can the TaqMan Cells-to-CT Express Kit be used for multiplexed qPCR?

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.
Are the components of the TaqMan Cells-to-CT Express Kit sold separately?

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.
Can I freeze and store cDNA generated with the TaqMan Cells-to-CT Express Kit?

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.
Can I freeze and store lysates from the TaqMan Cells-to-CT Express Kit?

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.
How do I identify genomic DNA (gDNA) contamination when using the TaqMan Cells-to-CT Express Kit?

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.

Does the TaqMan Cells-to-CT Express Kit contain reagents for the removal of genomic DNA (gDNA)?

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.

Which cell lines are compatible with the TaqMan Cells-to-CT Express Kit?

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.
Can I use the TaqMan Cells-to-CT Express Kit with larger microplate well sizes?

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.
Can I use the TaqMan Cells-to-CT Express Kit with both adherent and suspension cells?

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.
Can the TaqMan Cells-to-CT Express Kit protocol be automated?

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.
Using the TaqMan Cells-to-CT Express Kit, can I go directly from cells to RT-qPCR without having to purify RNA?

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

For Research Use Only.