A complete workflow for qPCR analysis from cultured cells

Cells-to-CT products, including 4 vials, 2 buffer bottles, and the kit box

Leading in gene expression analysis techniques, Invitrogen Cells-to-CT kits measure relative gene expression by real-time PCR—also known as quantitative PCR or qPCR—without RNA purification prior to amplification. Available in both TaqMan and SYBR Green detection chemistries, Cells-to-CT kits provide a complete workflow for qPCR analysis directly from cultured cells, providing you with excellent performance, reliability, and world-class support.

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Benefits of Cells-to-CT kits

  • Extraordinary ease and speed—96 samples for RT-qPCR in less than 10 min
  • Skip RNA purification—no columns, heating, or centrifugation
  • Exceptional performance—designed for consistent accuracy, reproducibility, and sensitivity with 1–100,000 cells
  • Analytically validated workflows—complete sets of reagents pre-optimized to work efficiently right out of the box

Selection guide: which Cells-to-CT kit is right for you?

 Detection chemistry availableTime to results (from cell lysis to qPCR results)Working rangeGene expression sensitivity
Cells-to-CT 2-step kitsSYBR Green* and TaqMan** formats2 hr10–100,000 cellsMedium to high levels of quantitation
Cells-to-CT 1-step kitsSYBR Green* and TaqMan** formats35–85 min10–100,000 cellsMedium levels of quantitation
Fast Advanced Cells-to-CT 2-step kitsSYBR Green* and TaqMan** formats80–95 min10–100,000 cellsMedium to high levels of quantitation
CellsDirect kitTaqMan** format only40–60 min1–10,000 cellsHigh levels of quantitation
Single Cell-to-CT kitTaqMan** format only3 hr1–10 cellsHigh levels of quantitation
* SYBR Green-based detection uses SYBR Green dye (a dsDNA-binding dye) to detect PCR product 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.
** TaqMan-based detection uses a fluorogenic probe specific to the target gene to detect the target as it accumulates during PCR. TaqMan-based detection generally has more sensitivity than SYBR Green-based detection.


1-step versus 2-step Cells-to-CT kits

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:

  • Have multiple targets quantified from cDNA
  • Need higher sensitivity for rare transcripts
  • Archive cDNA
  • Use liquid handling robotics

TaqMan format versus SYBR Green format

Choose the TaqMan format:

  • For time efficiency
  • For higher specificity and sensitivity
  • For multiplexing

Choose the SYBR Green format:

  • For cost efficiency
  • When using custom primers 

CellsDirect vs FastAdvanced Cells-to-CT kits vs traditional Cells-to-CT kits

CellsDirect: Real-time qPCR results directly from cells

  • Increased sensitivity—No RNA isolation steps, avoiding sample loss and PCR inhibition; use of SSIII for extensive sensitivity down to one cell/reaction
  • Significant savings—Less hands-on time and a lower cost than RNA purification kits
  • Robust qPCR results—Real-time detection of targets from a single cell to 10,000 cells
  • Versatility—Compatible with a wide variety of cell lines and samples from laser capture microdissection (LCM)

CellsDirect qPCR kits deliver highly sensitive and specific, real-time qPCR results directly from cells, without the need for an RNA purification step when testing with less than 10,000 cells per reaction to as low as one cell. CellsDirect technology is designed for maximum sensitivity with small samples (Figure 1).

 

CellsDirect 1-Step qPCR kits combine sensitivity with the highest level of convenience and throughput. With CellsDirect 1-step kits, just add your cell lysate directly to your qPCR reaction mixture, place it in your real-time instrument, and go. These kits are ideal for gene ex¬pression assays from samples collected using LCM (Figure 2).

Fast Advanced Cells-to-CT kits: Real-time qPCR results directly from cells

Skip RNA purification entirely with Fast Advanced Cells-to-CT Kits—going straight from cultured cell samples to measuring relative gene expression with real-time RT-PCR (RT-qPCR). 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.

Fast Advanced Cells-to-CT 2-step kits are faster and more convenient to use than traditional RNA purification without compromising sensitivity (Figure 3). 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 (Figure 4). 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 of TaqMan Fast Advanced Cells-to-CT kit 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).

Bar graphs of gene expression in HepG2 cells using the Fast Advanced Cells-to-CT kit compared to the traditional Cells-to-CT kit and a competing supplier

Figure 4. 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.

Cells-to-CT kits: Real-time qPCR results directly from cells

Cells-to-CT kits provide a complete workflow for real-time 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 probe-based (TaqMan probes) or dye-based (SYBR Green dye) master mixes for real-time PCR analysis.

All components of the Cells-to-CT Kits have been optimized for consistent and reliable performance. This helps minimize 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 5).

Figure 5. 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.

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 6).

Figure 6. Good sensitivity obtained in TaqMan gene expression assays with lysates generated using the Cells-to-CT Kit. 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.

CellsDirect: Real-time qPCR results directly from cells

  • Increased sensitivity—No RNA isolation steps, avoiding sample loss and PCR inhibition; use of SSIII for extensive sensitivity down to one cell/reaction
  • Significant savings—Less hands-on time and a lower cost than RNA purification kits
  • Robust qPCR results—Real-time detection of targets from a single cell to 10,000 cells
  • Versatility—Compatible with a wide variety of cell lines and samples from laser capture microdissection (LCM)

CellsDirect qPCR kits deliver highly sensitive and specific, real-time qPCR results directly from cells, without the need for an RNA purification step when testing with less than 10,000 cells per reaction to as low as one cell. CellsDirect technology is designed for maximum sensitivity with small samples (Figure 1).

 

CellsDirect 1-Step qPCR kits combine sensitivity with the highest level of convenience and throughput. With CellsDirect 1-step kits, just add your cell lysate directly to your qPCR reaction mixture, place it in your real-time instrument, and go. These kits are ideal for gene ex¬pression assays from samples collected using LCM (Figure 2).

Fast Advanced Cells-to-CT kits: Real-time qPCR results directly from cells

Skip RNA purification entirely with Fast Advanced Cells-to-CT Kits—going straight from cultured cell samples to measuring relative gene expression with real-time RT-PCR (RT-qPCR). 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.

Fast Advanced Cells-to-CT 2-step kits are faster and more convenient to use than traditional RNA purification without compromising sensitivity (Figure 3). 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 (Figure 4). 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 of TaqMan Fast Advanced Cells-to-CT kit 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).

Bar graphs of gene expression in HepG2 cells using the Fast Advanced Cells-to-CT kit compared to the traditional Cells-to-CT kit and a competing supplier

Figure 4. 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.

Cells-to-CT kits: Real-time qPCR results directly from cells

Cells-to-CT kits provide a complete workflow for real-time 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 probe-based (TaqMan probes) or dye-based (SYBR Green dye) master mixes for real-time PCR analysis.

All components of the Cells-to-CT Kits have been optimized for consistent and reliable performance. This helps minimize 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 5).

Figure 5. 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.

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 6).

Figure 6. Good sensitivity obtained in TaqMan gene expression assays with lysates generated using the Cells-to-CT Kit. 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.

Single Cell-to-CT kits

Single-cell analysis can be critical in applications such as candidate drug screening, cell differentiation and stem cell studies, and measuring individual cell responses to specific stimuli. Because tissues are composed of heterogeneous mixtures of cells, gene expression measurements based on the homogenized population don’t account for the small but critical changes occurring in individual cells.

This kit provides a validated workflow for gene expression analysis and offers a standardized platform for the study of single cells. 

Single-cell gene expression applications

Rare cells or events

  • Circulating metastatic cells
  • Fetal cells in maternal blood
  • Events within a library

Scarce, precious sample

  • Archival tissue
  • Clinical sample (fresh tumor)
  • Biomarker discovery

Single-cell precision in populations

  • Drug candidate research
  • Cell differentiation (e.g., stem cells)
  • Stochastic responses to stimuli

Real-time PCR applications for a complete experimental workflow solution

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.

Videos and webinars on Cells-to-CT kits

Cells-to-CT: full video

Learn how to prepare RNA for gene expression analysis from cultured cells, without the need for RNA purification. Fast track to RT-qPCR in only seven minutes. 

Cells-to-CT: PBS solution

Wash cells with PBS and aspirate PBS during the Cells-to-CT protocol.

Cells-to-CT lysis solution

Add the lysis solution to wells with cells, mix by pipetting up and down, and let stand for five minutes at room temperature during the Cells-to-CT protocol.

Cells-to-CT stop solution

Add the stop solution to the cells in lysis solution, and wait for two minutes at room temperature during the Cells-to-CT protocol.

Cells-to-CT RT master mix

Watch the preparation of the RT master mix during the Cells-to-CT protocol.

Cells-to-CT Kits: A fast way to kill your cells for gene expression analysis

This ASCB presentation shows you how Cells-to-CT kits allow you to measure relative gene expression by qPCR without having to purify RNA prior to amplification.

How to analyze gene expression from cultured cells

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

Less time to Real-Time PCR with Cells-to-CT kits

You get real-time PCR results in less time with the Cells-to-CT kits. They are fast, easy, and robust. Fewer handling steps help ensure shorter protocol time, increased consistency, minimal sample loss, and a reduced risk for contamination.

Frequently asked questions (FAQs) on Cells-to-CT kits

Cells-to-CT kits FAQs

Cells-to-CT kits provide a complete workflow for real-time qRT-PCR analysis directly from cultured cells without RNA purification. Reverse transcription and qPCR are performed in a single well. The kits use reverse transcription (RT) reagents for cDNA synthesis and are available with dye-based (SYBR Green dye) or probe-based (TaqMan probes) 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, reducing overall protocol time.

Based on the experiment on ten samples below, the Cells-to-CT kit produces 6.6 g of plastic waste and 0 mL: hazardous waste in seven minutes compared to 140 g plastic waste and 18 mL hazardous waste with standard RNA purification with RNeasy.

RNeasy
35 minutes
140 g plastic waste
18 mL hazardous waste
RNeasy reduces waste and hazardous materialsCells-to-CT
7 minutes
6.6 g plastic waste
0 mL hazardous waste
  1. Ensure all media is removed from the wells
  2. Wash with an equal volume of room temperature 1X PBS after the media removed
  3. Ensure reaction happens at room temperature (lysis reaction may not reach room temperature if plate is on ice, quickly moved to bench, or cold lysis solution is added)
  4. Warm lysis solution to room temperature before adding to cells
  5. Allow lysis reaction to proceed for 8 minutes
  6. Perform lysis reaction at 25°C for up to 8 minutes

Yes. Cells to CT kits are compatible with multiplex qPCR assays. If many targets are being tested, we recommend using the TaqMan format.

Cells-to-CT technology features a unique method for lysing cultured cells while removing genomic DNA (gDNA) and preserving RNA integrity. Therefore, by following the lysis/DNase treatment steps in the protocol, gDNA contamination will not be an issue for qPCR.

Single Cell-to-CT kits FAQs

Yes, it will be enough material for microarray analysis. The volume of the sample after the PreAmp step is 26.5 µL which is diluted 1:20 for a total volume of 530 µL, which is more than enough for a large number of TaqMan qPCR assays, TLDA cards or use in the QuantStudio open array.

While we have not tried this in-house, it is reason to believe this should work. However, optimization of the protocol around the RT step may be needed, as FFPE samples have a lot of RNA crosslinking which could affect the efficiency of the RT enzyme. Heating the sample for 10 min at 80°C before adding the RT in order to get good cDNA conversion should improve reverse transcription.

Cell lines compatible with Cells-to-CT kits

Cell lineGrowth typeSource speciesSource tissue
A549adherentH. sapiensLung carcinoma
BJadherentH. sapiensForeskin
CHO-K1adherentC. griseus (hamster)Ovary
COS-7adherentC. aethiops (monkey)Kidney
DU-145adherentH. sapiensProstate
HEK-293adherentH. sapiensKidney
HeLaadherentH. sapiensCervical adenocarcinoma
HepG2adherentH. sapiensLiver carcinoma
Huh-7adherentH. sapiensLiver
JurkatsuspensionH. sapiensAcute T cell leukemia
K562suspensionH. sapiensBone marrow
ME-180adherentH. sapiensCervical epidermoid carcinoma
NCI-H460adherentH. sapiensLung
Neuro 2AadherentM. musculusBrain
NIH/3T3adherentM. musculus (mouse)Embryonic fibroblast
PC-12adherentR. norvegicus (rat)Adrenal pheochromocytoma
Primary hepatocytesadherentH. sapiensLiver
PT-K75adherentS. scrofa (pig)Nasal turbinate mucosa
RajisuspensionH. sapiensB lymphocyte
SK-N-ASadherentH. sapiensBrain neuroblast
SK-N-SHadherentH. sapiensBrain fibroblast
U-2 OSadherentH. sapiensBone
U-87 MGadherentH. sapiensBrain glioblastoma 

Note: Due to differences in cell size and composition, the maximum number of cells per lysis reaction may be slightly different for different cell lines. Test for inhibition and minimal sample input by using the TaqMan Cells-to-CT Control Kit.

Cells-to-CT kit ordering information
Resources
  • Protocol Videos—Videos to help you isolate nucleic acids using a variety of techniques.
  • DNA & RNA Selection Guide—Find the right purification products.
  • Videos—View our library of DNA & RNA purification and analysis videos.
  • Application Notes—Explore our application notes from scientists sharing data for isolation products

For Research Use Only. Not for use in diagnostic procedures.