Ambion Cells-to-CT Kits

As an inovative leader in gene expression analysis techniques, Invitrogen Cells-to-CT Kits allow you to measure relative gene expression by real-time RT-PCR without having to purify RNA prior to amplification. Cells-to-CT Kits are available with TaqMan and SYBR Green detection chemistries. Our Cells-to-CT kits provide a complete workflow for real-time qRT-PCR analysis directly from cultured cells enabling you to get true value with excellent performance, reliability, and world-class support.

  • Extraordinary ease and speed—96 samples for qRT-PCR in typically <10 min
  • No tedious RNA purification—no columns, heating, or centrifugation
  • Superior performance—designed for consistent accuracy, reproducibility, and sensitivity with 1–100,000 cells
  • Validated kit workflows—complete sets of reagents preoptimized to work efficiently right out of the box

Download guide What’s new

1-step or 2 step qRT-PCR?

Choose 1-step if you:

  • Have many samples with one or a few targets
  • Do not store cDNA
  • Use liquid handling robotics

Choose a 2-step kit if you:

  • You need higher sensitivity for rare transcripts
  • Archiving of cDNA is possible
  • Multiple targets will be quantified from cDNA
  • You use liquid handling robotics

More on choosing a 1- or 2-step RT-PCR kit


TaqMan or SYBR Green chemistry?

TaqMan detection typically offers a shorter protocol, higher specificity and sensitivity, and the ability to multiplex. SYBR Green dye–based methods are often regarded as more flexible in terms of the range of primers that can be used, and the cost is typically lower.

More on choosing a detection method


Cells-to-CT Kit selection guide

  Fast Advanced Cells-to-CT kits Cells-to-CT kits
One complete workflow Lysis → RT → qPCR
Cells-to-CT Lysis, DNase, and Stop Solutions Yes
Detection chemistry SYBR Green format* TaqMan format** SYBR Green format* TaqMan format**
Time to results 95 min 80 min 120 min
Working range 10–100,000 cells 10–100,000 cells
Specificity Medium† High Medium† High
Sensitivity (maximum percentage of sample that makes it into the qPCR reaction) 13.5% lysate 20% lysate 13.5% lysate 20% lysate
Reproducibility Medium† High Medium† High
Multiplexing        
Predesigned assays        
Typically requires user design, experimental optimization        
Gene expression Medium level of quantitation High level of quantitation Medium level of quantitation High level of quantitation
Applications
  • Gene expression
  • DNA quantitation
  • CHiP
  • SNP genotyping
  • Copy number variation
  • Pathway analysis
  • microRNA and small RNAs
  • Mutation detection
  • Multiplexing
  • Gene expression
  • DNA quantitation (pathogen detection)
  • CHiP
  • Gene expression
  • DNA quantitation
  • CHiP
  • SNP genotyping
  • Copy number variation
  • Pathway analysis
  • microRNA and small RNAs
  • Mutation detection
  • Multiplexing
Detection
  • SYBR Green primers
  • SYBR Green master mixes
Order Now
  • TaqMan Assays
  • TaqMan master mixes

Order Now
  • SYBR Green primers
  • SYBR Green master mixes
Order Now
  • TaqMan Assays
  • TaqMan master mixes

Order Now

* SYBR Green–based detection uses SYBR Green dye (a dsDNA-binding dye) to detect PCR product as it accumulates during PCR.
** TaqMan-based detection uses a fluorogenic probe specific to the target gene to detect the target as it accumulates during PCR.
† SYBR Green–based assays require primers to be designed and validated to ensure specificity. Specificity and reproducibility depend on the template quality and primer design and optimization.

  Order Now Order Now
  Cells-to-C1-Step TaqMan Kit Cells-to-CT 1-Step Power SYBR Green Kit
  1-step qPCR sample prep 1-step qPCR sample prep
Detection chemistry TaqMan SYBR Green
Specificity High Medium*
No. of cells in lysis step 10–100,000 cells 10–100,000 cells
Detection sensitivity 1–10 copies Variable*
Reproducibility High Medium*
RNA purification required No No
Sample prep time (to qRT-PCR) for 8 samples 7 min 7 min
Total time (from cell lysis to qRT-PCR results) 35 min 1 hr 23 min
Multiplexing    
cDNA archiving    
Includes RT enzyme and master mix    
Includes preamplification reagents    
Suitable for automation    
Flexibility Medium Medium
  Order Now
  Single Cell-to-CqRT-PCR Kit
  Single Cell-to-CT Kit
Detection chemistry TaqMan
Specificity High
No. of cells in lysis step 1–10 cells
Detection sensitivity 1–10 copies
Reproducibility High
RNA purification required No
Sample prep time (to qRT-PCR) for 8 samples 7 min
Total time (from cell lysis to qRT-PCR results) 3 hr
Multiplexing  
cDNA archiving  
Includes RT enzyme and master mix  
Includes preamplification reagents  
Suitable for automation  
Flexibility High

* Depends on template quality and primer/design optimization.

  Fast Advanced Cells-to-CT kits Cells-to-CT kits
One complete workflow Lysis → RT → qPCR
Cells-to-CT Lysis, DNase, and Stop Solutions Yes
Detection chemistry SYBR Green format* TaqMan format** SYBR Green format* TaqMan format**
Time to results 95 min 80 min 120 min
Working range 10–100,000 cells 10–100,000 cells
Specificity Medium† High Medium† High
Sensitivity (maximum percentage of sample that makes it into the qPCR reaction) 13.5% lysate 20% lysate 13.5% lysate 20% lysate
Reproducibility Medium† High Medium† High
Multiplexing        
Predesigned assays        
Typically requires user design, experimental optimization        
Gene expression Medium level of quantitation High level of quantitation Medium level of quantitation High level of quantitation
Applications
  • Gene expression
  • DNA quantitation
  • CHiP
  • SNP genotyping
  • Copy number variation
  • Pathway analysis
  • microRNA and small RNAs
  • Mutation detection
  • Multiplexing
  • Gene expression
  • DNA quantitation (pathogen detection)
  • CHiP
  • Gene expression
  • DNA quantitation
  • CHiP
  • SNP genotyping
  • Copy number variation
  • Pathway analysis
  • microRNA and small RNAs
  • Mutation detection
  • Multiplexing
Detection
  • SYBR Green primers
  • SYBR Green master mixes
Order Now
  • TaqMan Assays
  • TaqMan master mixes

Order Now
  • SYBR Green primers
  • SYBR Green master mixes
Order Now
  • TaqMan Assays
  • TaqMan master mixes

Order Now

* SYBR Green–based detection uses SYBR Green dye (a dsDNA-binding dye) to detect PCR product as it accumulates during PCR.
** TaqMan-based detection uses a fluorogenic probe specific to the target gene to detect the target as it accumulates during PCR.
† SYBR Green–based assays require primers to be designed and validated to ensure specificity. Specificity and reproducibility depend on the template quality and primer design and optimization.

  Order Now Order Now
  Cells-to-C1-Step TaqMan Kit Cells-to-CT 1-Step Power SYBR Green Kit
  1-step qPCR sample prep 1-step qPCR sample prep
Detection chemistry TaqMan SYBR Green
Specificity High Medium*
No. of cells in lysis step 10–100,000 cells 10–100,000 cells
Detection sensitivity 1–10 copies Variable*
Reproducibility High Medium*
RNA purification required No No
Sample prep time (to qRT-PCR) for 8 samples 7 min 7 min
Total time (from cell lysis to qRT-PCR results) 35 min 1 hr 23 min
Multiplexing    
cDNA archiving    
Includes RT enzyme and master mix    
Includes preamplification reagents    
Suitable for automation    
Flexibility Medium Medium
  Order Now
  Single Cell-to-CqRT-PCR Kit
  Single Cell-to-CT Kit
Detection chemistry TaqMan
Specificity High
No. of cells in lysis step 1–10 cells
Detection sensitivity 1–10 copies
Reproducibility High
RNA purification required No
Sample prep time (to qRT-PCR) for 8 samples 7 min
Total time (from cell lysis to qRT-PCR results) 3 hr
Multiplexing  
cDNA archiving  
Includes RT enzyme and master mix  
Includes preamplification reagents  
Suitable for automation  
Flexibility High

* Depends on template quality and primer/design optimization.

New Invitrogen Fast Advanced Cells-to-CT  kits offer researchers the ability to skip RNA purification entirely by going straight from cultured cell samples to measuring relative gene expression by real-time RT-PCR (RT-qPCR). And for flexibility, Cells-to-CT kits are available in both TaqMan and SYBR Green formats. These kits provide a complete workflow for RT-qPCR analysis directly from cultured cells without RNA purification. Featuring a unique method for lysing cultured cells while removing genomic DNA (gDNA) and preserving RNA integrity, the kits contain reverse transcription (RT) reagents for cDNA synthesis and either TaqMan or SYBR Green master mixes for real-time PCR analysis.

Two formats available for increased flexibility

The new TaqMan Fast Advanced Cells-to-CT Kit utilizes an improved reverse transcriptase enzyme that is more sensitive to detect abundant or rare transcripts. This makes it possible to perform expression analysis directly from cultured cells without RNA purification. This kit helps save time and offers a simple workflow that is suitable for a few samples, but can also be easily incorporated into automated, high-throughput applications.

The TaqMan Fast Advanced Cells-to-CT Kit offers:

  • The same proprietary Lysis Solution and Stop Solution as the original TaqMan Gene Expression Cells-to-CT Kit
  • Superior specificity for detecting targets
  • Extraordinary ease and speed—96 samples for RT-qPCR in typically <10 min
  • No tedious RNA purification—no columns, heating, centrifugation, or sample transfer
  • Superior performance—designed for consistent accuracy, reproducibility, and sensitivity with 10–100,000 cells
  • Premium validated kit workflows—complete sets of reagents preoptimized to work efficiently right out of the box
  • Detect virtually any gene product with more than 2 million predesigned assays
  • Affordable assays for nearly every human, mouse, and rat gene in the RefSeq database
  • Available for 28 species and some pathogens

The new SYBR Green Fast Advanced Cells-to-CT Kit is a fast, easy, and robust method to go directly from cultured cells to real- time PCR results. This proven cell lysis and RNA stabilization technology eliminates the laborious and time-consuming RNA purification process completely. The kit is powered by Applied Biosystems AmpliTaq Gold DNA Polymerase, LD (Low DNA), which provides the highest levels of specificity with standard real-time PCR.

The SYBR Green Fast Advanced Cells-to-CT Kit offers:

  • The same proprietary Lysis Solution and Stop Solution as the original TaqMan Gene Expression Cells-to-CT Kit
  • High specificity for detecting targets
  • Extraordinary ease and speed—96 samples for RT-qPCR in typically <10 min
  • No tedious RNA purification—no columns, heating, or centrifugation
  • Superior performance—designed for consistent accuracy, reproducibility, and sensitivity with 10–100,000 cells
  • Productive validated kit workflows— complete sets of reagents preoptimized to work efficiently right out of the box

Higher sensitivity in less time

The Cells-to-CT Stop Solution allows for greater sample input in the downstream RT and qPCR reactions, greatly increasing the sensitivity for detecting rare targets. Both TaqMan and SYBR Green Fast Advanced Cells-to-CT Kits use the Stop Solution, providing excellent sensitivity with a very fast workflow. These kits are approximately 48 times faster than most high-throughput RNA purification kits. Fast Advanced Cells-to-CT kits can process 96 or 384 wells at once; the experimental workflow only takes about 10 minutes for a 96- or 384-well plate. Products from other suppliers use low pH and dilution in their workflows, which restricts how much cell lysate can be used in downstream steps.

Gene expression experimental workflow: comparison

Figure 1. Volume of lysate or RT reaction at each step in the workflow. Real-time ready kits are the main competition for our current Cells-to-CT portfolio.

Gene expression experimental workflow

Figure 2. The procedure for Fast Advanced Cells-to-CT kits is a 2-step process that involves reverse transcription and qPCR following cell lysis. A 1-step reaction is when RT and qPCR are performed in a single well. In the qPCR step, TaqMan Assays use custom primer and probe combinations designed for each target. These assays are more sensitive and specific than SYBR Green assays, which use generic qPCR primers.

Cells-to-CT kits provide a complete workflow for real-time qRT-PCR analysis directly from cultured cells without RNA purification. Featuring a unique method for lysing cultured cells while removing genomic DNA (gDNA) and preserving RNA integrity, the kits contain reverse transcription (RT) reagents for cDNA synthesis and either probe-based (TaqMan probes) or dye-based (SYBR Green dye) master mixes for real-time PCR analysis.

The 1-step and 2-step kits differ in that the 1-step kits combine reverse transcription and real-time PCR into a single step, significantly reducing the number of pipetting steps and protocol time.

Whether you are using plates or tubes, the Cells-to-CT Kits use a simple 7–8 minute sample preparation procedure outlined below (Figure 2). The lysis technology is designed for 10–100,000 cultured cells per sample. Cells are washed in PBS, then lysed for 5 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 analysis on your real-time PCR instrument, or they can be stored at –20°C for up to 5 months. The 2-step kit workflow proceeds with reverse transcription to synthesize cDNA, after which the cDNA samples can be either archived or analyzed directly by real-time qPCR.

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 traditional 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 7–8 minutes of reaction time, allowing complete cell culture processing from sample to qRT-PCR answer in as little as 35 minutes (e.g., Cells-to-CT 1-Step TaqMan Kit).

Fast Advanced Cells-to-CT 2-step kits are faster and more convenient to use than traditional RNA purification without compromising sensitivity. Because the workflow is lysate-based, there is no loss of RNA with Cells-to-CT kits. In traditional RNA purification, a small amount of RNA is always lost to incomplete elution off of filters or magnetic beads. Thus, the ability to detect rare RNAs in low input cell samples (10–1,000) is higher when samples are processed with Cells-to-CT kits compared to results from the same samples using RNA purification workflows. This, coupled with the benefits of the Stop Solution, gives Cells-to-CT workflows more sensitivity than competitor kits to detect both abundant and low copy transcripts.

Figure 3. Sensitivity compared to other kits. 104 cells were lysed following each kit’s protocol. The maximum amount of lysate was added to each kit’s RT (45% for Cells-to-CT and Fast Advanced Cells-to-CT kits; 10% for other supplier). Then 25% of RT was added into the qPCR reactions of a TaqMan Array for phosphodiesterases, using each kit’s qPCR master mix. The Fast Advanced Cells-to-CT kit performed on average 0.5 Ct better than the original Cells-to-CT kit and 3.6 Ct better than another supplier’s kit (11x more sensitive than other supplier).

Figure 4. Sensitivity compared to other kits. 104 cells were lysed following each kit’s protocol. The maximum amount of lysate was added to each kit’s RT (45% for the original Cells-to-CT kit and Fast Advanced Cells-to-CT kit; 10% for the other supplier’s kit). Then 25% of RT was added into the qPCR reactions of a TaqMan Array for phosphodieterases, using each kit’s qPCR master mix. Conclusions: The Fast Advanced Cells-to-CT kit performs on average 0.7 Ct better than the original Cells-to-CT kit and 4.7 Ct better than the other supplier’s kit. This means that the Fast Advanced kit is 14x more sensitive than the other supplier’s kit.

Figure 5. Comparison of detection. 104 HepG2 cells were lysed following each kit’s protocol. The maximum amount of lysate was added to each kit’s RT (45% for Cells-to-CT and Fast Advanced Cells-to-CT kits; 10% for other supplier). Then 25% of RT was added into the qPCR reactions for the other supplier’s kit; 30% was added to the qPCR reaction for the Fast Advanced Cells-to-CT kit, 45% added to Power SYBR kit. Assayed 10 different genes. The majority of genes were not detected in this cell line. Of the genes that were detected, the Fast Advanced Cells-to-CT kit has lower Ct values than the other kits for most genes.

Fast Advanced Cells-to-CT kits include a sample preparation step that can be completed in less than 10 mins for 96 or 384 samples. High throughput RNA isolation methods like magnetic beads kits can process 384 samples, but with a protocol duration of 3 hours*. Considering that in a typical drug target validation screen, multiple 96 or 384 well plates are processed, we compared the time needed to prepare 1536 samples (i.e. 4 x 384 plates or 16 X 96 well plate).

  • Cells-to-CT takes about 40 mins to 1 hour for 1536 samples (i.e. 4 plates of 384 well format).
  • Bead Based Isolation Protocol takes about 48 hours for 1536 samples (i.e. 16 plates of 96 well format, each plate takes 3 hours).
  • Cells-to-CT kits are 48X faster.

* based on the protocol duration of MagMAX mirVana Total RNA Isolation Kit

All components of the Cells-to-CT Kits have been optimized for consistent and reliable performance. This minimizes the guesswork involved in assembling separate sample preparation and real-time RT-PCR master mixes.

For added quality assurance, the Cells-to-CT 1-Step TaqMan Kit has been validated with TaqMan Gene Expression Assays, and the Cells-to-CT 1-Step Power SYBR Green Kit has been validated with a set of primers for common gene targets. Both show performance equivalent to that obtained with purified RNA (Figure 6).

The performance of the TaqMan Gene Expression Cells-to-CT Kit was compared to other commercially available lysis kits and to purified RNA. Inputs of 100–100,000 cells per lysis reaction were examined. The sensitivity of the TaqMan Cells-to-CT Kit protocol was equivalent to that obtained with purified RNA, and it surpassed those from other suppliers (Figure 7).

Figure 6. Sensitivity and detection of limited target sequences using Cells-to-CT Kits. (A) Sensitivity of gene expression assays with lysate vs. purified RNA. HeLa cells (10–100,000) were processed in triplicate using the Cells-to-CT 1-Step TaqMan Kit, or RNA was purified using an RNA spin column method. Each set of samples was analyzed by real-time RT-PCR on the 7900HT Real-Time PCR System, for XENO and ACTB from the Cells-to-CT Control Kit. (B) Detection of limited target sequences. Increasing amounts of NIH3T3 cells (mouse) were added to 10,000 HeLa cells. The cells were processed in triplicate using the TaqMan Gene Expression Cells-to-CT Kit (2-step procedure) or purified reagents with an RNA spin column method. All samples were reverse transcribed for mouse-specific (B2M) and human-specific (TKT) genes in triplicate reactions on a 7900HT Fast Real-Time PCR System.

Figure 7. Good sensitivity obtained in TaqMan Gene Expression Assays with lysates generated using the Cells-to-CKit. HeLa cells (100–100,000) were prepared using either traditional RNA purification, the TaqMan Gene Expression Cells-to-CT Kit, or other lysate methods from suppliers Q and S. Reverse transcription reactions were made with the maximum recommended sample input for each kit, and real-time PCR was performed in triplicate using a PPIA TaqMan Gene Expression Assay.

To help you experience the power of optimized results across diverse applications, we also offer real-time PCR instrumentation solutions to complete your experimental workflow. Real-time PCR is used for sensitive, specific detection and quantification of nucleic acid targets. We have developed powerful assay design algorithms, optimized master mixes, intuitive data analysis software, and flexible instrumentation to help harness the power of qPCR across a rich and diverse set of applications. Discover solutions for your qPCR-based research.

Find out more at thermofisher.com/quantstudioqpcrfamily

Cells-to-CT Kits Reviews, Videos & Webinars

Read what scientists have to say about the Cells-to-CT Kits

The TaqMan Gene Expression Cells-to-CT Kit has performed consistently. The performance is increased as compared to RNA purification, RT steps, and then qRT-PCR. There is less handling, less chance at mistakes. Because of this time savings and increase in sensitivity, I’ve incorporated this protocol into the Human Embryonic Stem Cell Training Course at the Stem Cell Research Center at Rutgers University.
     
 

—Director, Stem Cell Core Facility

 
 
 
 
Gene expression quantification on cultured cells using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) typically involves an RNA purification step that limits sample processing throughput and precludes parallel analysis of large numbers of samples. An approach in which cDNA synthesis is carried out on crude cell lysates instead of on purified RNA samples can offer a fast and straightforward alternative. Here, we evaluate such an approach, benchmarking the Ambion Cells-to-CT kit with the classic workflow of RNA purification and cDNA synthesis, and demonstrate its good accuracy and superior sensitivity.
     
 

—Van Peer G, Mestdagh P, Vandesompele J (2012) Accurate RT-qPCR gene expression analysis on cell culture lysates. Sci Rep 2:222.

 
Videos

How to analyze gene expression from cultured cells

Learn how to prepare RNA for gene expression analysis from cultured cells in 7 minutes.

Webinar

Single cell gene expression profiling in neural stem cells–innovative solutions for sample prep and analysis

Read what scientists have to say about the Cells-to-CT Kits

The TaqMan Gene Expression Cells-to-CT Kit has performed consistently. The performance is increased as compared to RNA purification, RT steps, and then qRT-PCR. There is less handling, less chance at mistakes. Because of this time savings and increase in sensitivity, I’ve incorporated this protocol into the Human Embryonic Stem Cell Training Course at the Stem Cell Research Center at Rutgers University.
     
 

—Director, Stem Cell Core Facility

 
 
 
 
Gene expression quantification on cultured cells using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) typically involves an RNA purification step that limits sample processing throughput and precludes parallel analysis of large numbers of samples. An approach in which cDNA synthesis is carried out on crude cell lysates instead of on purified RNA samples can offer a fast and straightforward alternative. Here, we evaluate such an approach, benchmarking the Ambion Cells-to-CT kit with the classic workflow of RNA purification and cDNA synthesis, and demonstrate its good accuracy and superior sensitivity.
     
 

—Van Peer G, Mestdagh P, Vandesompele J (2012) Accurate RT-qPCR gene expression analysis on cell culture lysates. Sci Rep 2:222.

 
Videos

How to analyze gene expression from cultured cells

Learn how to prepare RNA for gene expression analysis from cultured cells in 7 minutes.

Webinar

Single cell gene expression profiling in neural stem cells–innovative solutions for sample prep and analysis

Resources

Product literature

Tips & tricks

Posters and papers

Application notes

Frequently asked questions