qPCR is used to quantify the number of copies of a given DNA sequence using PCR.
qPCR is short for “quantitative polymerase chain reaction”.
PCR is the process of exponentially amplifying DNA.
qPCR adds fluorescent dyes and fluorometers to the standard PCR process. qPCR quantifies the target in a given sample in real time by showing the presence of nucleic acid sequences from the PCR process. With qPCR, in “real-time,” fluorophores are incorporated into the amplified product and the amount of fluorescence can be detected and monitored.
qPCR is sometimes referred to as real-time PCR, but it should never be confused with RT-PCR. Reverse transcription polymerase chain reaction (RT-PCR) is different from qPCR because it uses RNA as the nucleic acid starting template.
qPCR is used to detect and quantify nucleic acids in a given sample. Because of its sensitivity, molecular biologists use qPCR to most popularly used to measure gene expression to get a better understanding of various diseases and other biological pathways. Other applications developed using qPCR include next-generation sequencing library quantification, pathogen detection, SNP detection, and microRNA detection and profiling.
The sequences with the adapter sequences serve as templates during the amplification process. A bonus of using qPCR is the minimal amount of material used and its ability to be automated. Both qualities allow for more high-throughput runs to be performed.
Keep in mind the data generated by qPCR runs is very dependent on the dyes and primers you use. If you use fluorescent DNA probes instead of dyes, you’ll be able to measure multiple DNA targets using multiplex qPCR.
Read this app note to learn how to develop multiplex qPCR assays.
During the qPCR process, the targeted DNA sequence is amplified and fluorescence is incorporated simultaneously. Each amplification cycle, the fluorescence is detected, measured, and plotted on a graph known as an amplification curve (Figure 1). The amplification cycle is automatically run and analyzed by a qPCR machine.
Traditionally, a threshold model of analysis has been used for qPCR to account for background fluorescence. However with updated technology, most qPCR software performs baseline correction and background subtraction that eliminates background noise. Therefore, the most robust method for analyzing qPCR data is regression mode. This method analyzes the fluorescence curve for each well to determine the point where the amplification becomes exponential.
To quantify the amount of target DNA or RNA in the sample, a standard curve is generated using known concentrations of a reference molecule and then the target DNA can be compared to the standard curve.
The Azure Cielo is a qPCR machine designed with special innovative optical technology. The Cielo has two sets of 16 optical fibers that allow 16 individual wells to be imaged simultaneously, meaning an entire 96-well plate can be imaged in up to six fluorescent channels in just nine seconds saving time and getting results quicker. This ultimately increases high-throughput and allow for more libraries to be quantified and faster.
Relative quantitation in qPCR is a powerful method for comparing gene expression levels among different samples. Instead of measuring exact quantities, how much a target gene’s expression changes relative to a reference sample, like a control, can be gauged.
The Delta Ct (ΔCt) is the difference between the Ct values of the target gene and the reference gene. To calculate the ΔCt, you need the Ct value. Using ΔCt, the fold change in gene expression between different samples can be calculated. This number indicates whether the target gene is upregulated or downregulated compared to the reference condition.
A qPCR workflow (Figure 2) typically involves several key steps to accurately quantify the amount of a specific DNA or RNA target in a sample.
Here are the fundamental steps in a qPCR workflow:
Primer and Probe Design
Master Mix Preparation
Calculation of Results
The sample workflow above may be adapted to your specific research needs, and variations may exist depending on the particular qPCR method being used (e.g., SYBR Green or TaqMan probe-based qPCR).
Watch this webinar that breakdowns the qPCR assay and teaches you how a good experimental design can reduce biological variability before the run here >>
Selecting the perfect qPCR machine that suits your research needs is a crucial decision, and can significantly impact the quality of your data. That’s where the Cielo shines. It’s designed to deliver reproducible and precise results, all while speeding up your run times so you can collect data in less time.
Imagine having up to six channels at your disposal, enabling you to detect up to six targets in each sample through multiplexing. The Cielo‘s smart use of fiber optics directly delivers light into each well, minimizing background fluorescence and ensuring outstanding specificity and precision. With the ability to capture approximately 100,000 pixels per well, the Cielo proves to be both sensitive and accurate, making it the ideal companion for your real-time PCR needs.
Looking for a standout qPCR machine? Azure is confident you will be impressed with the ease of use and performance of the Cielo has to offer. We’ll arrange to send your lab a Cielo to use for one week, without any obligation, absolutely free. Sign up for the trial program now and be on your way to better data.
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