Expedite gene expression for your RT-qPCR

What is Real-Time Quantitative PCR and how does a lysate-based system expedite gene expression?

Real-Time Quantitative PCR terminology

Real-Time reverse transcription polymerase chain reaction, often abbreviated RT-qPCR, is a technique used to detect and quantify RNA. It is particularly useful for measuring gene expression levels. But how did this nomenclature come to pass?

Let’s start with the basics. A Polymerase Chain Reaction, or PCR, is a widely used molecular biology technique for amplifying and creating more copies of specific segments of DNA. This process allows scientists to analyze DNA in detail, even when the initial sample is very small.

Endpoint PCR: Absence or presence

Bulk PCR, also known as endpoint PCR, is the most traditional form of PCR. It is primarily used to determine the presence or absence of a specific DNA sequence. The process involves amplifying the DNA and then analyzing the endpoint data to provide a simple “Yes/No” answer regarding the target of interest. This method is straightforward and effective for applications where qualitative data is sufficient.

Key Features:

• Provides a “Yes/No” answer.
• Suitable for detecting the presence of specific DNA sequences.
• Does not provide quantitative data.

Quantitative PCR (qPCR): Real-Time Measurements

Quantitative PCR, or qPCR, takes PCR a step further by allowing for the real-time measurement of DNA amplification. This technique requires the use of a standard curve to provide relative quantitative data. By monitoring the amplification process in real-time, qPCR can quantify the amount of DNA present in the sample, making it invaluable for applications such as gene expression analysis and viral load quantification.

Key Features:

• Provides real-time measurements of DNA amplification.
• Requires a standard curve for relative quantitation.
• Useful for quantifying gene expression

Digital PCR (dPCR): Absolute Quantitation

Digital PCR (dPCR) is a more recent advancement in PCR technology that offers absolute quantitation of known targets. Unlike qPCR, dPCR does not require a standard curve, which enhances its precision and reproducibility. By partitioning the sample into thousands of individual reactions, dPCR can provide a highly accurate count of DNA molecules, making it ideal for applications requiring precise quantitation, such as rare mutation detection and copy number variation analysis.

Key Features:

• Provides absolute quantitation without the need for a standard curve.
• Offers greater precision and reproducibility compared to qPCR.
• Ideal for detecting rare mutations and analyzing copy number variations.

Figure 1a.

Figure 1b.

Figure 1c.

Workflow from sample to PCR amplification

In standard workflows, nucleic acid is isolated from a sample which can often take time and manual effort depending on the type of isolation method (i.e. chemical precipitation, spin columns, or magnetic bead-based isolation). Utilizing automation techniques with magnetic bead chemistries may enable a faster turnaround time compared to manual methods like precipitation or spin columns. MagMAX™ kits and and Dynabeads™ or Pierce™ chemistries on automated platforms like the KingFisher™ instruments can provide easy to use, higher throughput capabilities to isolate such nucleic acids, cells, or proteins but they still require investment in the technology and consumables. Although isolation of nucleic acid can significantly be improved in terms of turnaround time, the time for processing magnetic bead-based workflows on typical benchtop instruments can take 30-45 minutes before heading into any downstream analytical evaluation of the nucleic acid or amplification of the isolated material with various PCR platforms which can add additional time to any workflow. Nucleic acid isolation with magnetic beads provides versatile and customizable solutions that can be scalable in any workspace with high-quality and pure nucleic acid.

Isolating nucleic acid is time-consuming, labor intensive, and may provide opportunity for sample degradation. Nucleic acid quantitation is prone to errors from machines or pipette miscalibration, or dilution. In addition, spectrophotometry cannot be used to detect RNA from low cell inputs. Using a fluorescent dye increases sensitivity up to 100–fold but can be expensive. There is a need for a faster turnaround time to PCR amplification to simplify some workflows in the field of pharma research for biomarker discovery and gene expression research.

Simplify relative quantitation using lysate-based RT-qPCR technology

With advances in technology, new solutions have been developed that enable real-time PCR of cultured cell samples without the need to isolate RNA. Potential problems such as sample mix-up and RNA degradation are minimized, and samples are not subject to RNA quantitation errors. With this type of lysate-based technology, the RNA within cells can be ready for reverse transcription (RT) in as little as 5-minutes. Samples can contain 10–100,000 cells, and prepared lysates can be used in RT reactions without quantitation of any kind.

Benefits of Lysate-Based Technology

1. Time Savings: Traditional RNA isolation can be a time-consuming process, often taking hours to complete. In contrast, the Cells-to-CT kits allow for RNA preparation in as little as 5 minutes. This rapid preparation is particularly beneficial in high-throughput settings where time efficiency is critical.
2. Ease of Use: The simplified workflow reduces the number of steps and the potential for errors. By eliminating the RNA isolation step, the risk of RNA degradation and sample mix-up is minimized, ensuring more reliable and reproducible results.
3. Direct Use in RT Reactions: The prepared lysates can be directly used in reverse transcription reactions without the need for RNA quantitation. This not only saves time but also reduces the variability introduced by quantitation errors.

	TaqMan™ Cells-to-CT™ Express Kit	TaqMan™ Cells-to-CT™ HepatoExpress Kit	Cells-to-CT™ 1-Step TaqMan kit	Single Cell-to-CT™ qRT-PCR Kit	TaqMan™ Fast Advanced Cells-to-CT™ Kit
	Title: Cells-to-CT Express Alt text: Cells-to-CT Express kit	Title: Cells-to-CT HepatoExpress Alt text: Cells-to-CT HepatoExpress kit	Title Cells-to-CT™ 1-Step TaqMan kit Alt text: Cells-to-CT™ 1-Step TaqMan kit	Title: Single Cell-to-CT Alt text: Single Cell-to-CT Kit	Title: Taq Man Fast Advanced Alt text: TaqMan Cells-to-CT Fast Advanced Kit
Cell working range	10-100,000 cells	≤ 10,000 cells	10-100,000 cells	1-10 cells	10-100,000 cells
1-step or 2-step	2-step	2-step	1-step	1-step	2- step
Sample Type	Mammalian cells	Primary Hepatocytes	Mammalian cells	Mammalian cells	Mammalian cells, 3D cell culture

The Cells-to-CT product line was designed to streamline the workflow for gene expression analysis by eliminating the need for RNA isolation. This innovative approach leverages lysate-based technology to prepare RNA directly from cultured cells, significantly reducing the time and complexity associated with traditional RNA isolation methods. There are a variety of Cells-to-Ct products including those with TaqMan or SYBR chemistries.

The latest addition to the Cells-to-CT product line is the TaqMan™ Cells-to-CT™ HepatoExpress[LM1]™ kit. This specialized kit is designed for hepatocyte cell samples, providing a tailored solution for researchers working with liver cells. The Hepato-Express Cells-to-CT kit offers the same benefits as the original Cells-to-CT kits, with optimized reagents and protocols for hepatocyte samples. This opens the door for gene expression research in primary hepatocytes for infectious disease and oncology research, drug metabolism and pharmacokinetics by expression, toxicology research, and stem cell research. This poster showcases how the Hepato-express Cells-to-CT kit can support CYP induction studies.

The entire workflow for Cells-to-Ct Hepato-Express from sample to answer takes 75 minutes and may be faster than most bead-based extraction workflows (sometimes taking up to 2 hours from sample to amplification), Spin-column workflows (sometimes taking up to 3 hours with extensive hands-on time), or luminescence assays (sometimes taking up to 4 hours or more with extensive hands on steps) which may significantly save time and increase productivity in gene expression research and biomarker discovery.

To learn more about this type of technology please see further resources at thermofisher.com/cellstoct

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

© 2026 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.