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Real-time quantitative PCR (qPCR)
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Real-time Quantitative PCR (qPCR) is a technique that involves adding fluorescent groups (including fluorescent dyes or fluorescently labeled specific probes) to the PCR reaction system, which can specifically bind to the DNA products. This allows for the marking and tracking of the PCR products. As the PCR reaction progresses, the reaction products accumulate, and the fluorescence signal intensity also increases proportionally. Every cycle, the fluorescence intensity signal is collected, enabling real-time monitoring of the change in product quantity throughout the PCR process. Combined with corresponding software, the products can be analyzed, resulting in a fluorescence amplification curve. This curve can be used to calculate the initial template quantity of the target gene in the sample being tested, and is commonly used to analyze changes in the expression level of the target gene.
INSTRUCTION & COA：
- Product Description
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Technical Principle
Real-time Quantitative PCR (qPCR) is a technique that involves adding fluorescent groups (including fluorescent dyes or fluorescently labeled specific probes) to the PCR reaction system, which can specifically bind to the DNA products. This allows for the marking and tracking of the PCR products. As the PCR reaction progresses, the reaction products accumulate, and the fluorescence signal intensity also increases proportionally. Every cycle, the fluorescence intensity signal is collected, enabling real-time monitoring of the change in product quantity throughout the PCR process. Combined with corresponding software, the products can be analyzed, resulting in a fluorescence amplification curve. This curve can be used to calculate the initial template quantity of the target gene in the sample being tested, and is commonly used to analyze changes in the expression level of the target gene.
Figure: Working Principle of SYBR Green Method
Technical advantages
Using the Roche LightCycler® 96 real-time fluorescence quantitative PCR instrument, it features high sensitivity, high repeatability, high resolution, a wide dynamic range, and stable performance. The data can accurately reflect the relative content changes of RNA in the samples.
2. Diversity of internal reference genes: For different species materials, different internal reference design schemes are provided. It is not always necessary to use GAPDH or β-actin. A scientific internal reference design scheme is more conducive to improving the reliability of fluorescence quantitative PCR data.
3. The reagent and consumable materials used in the fluorescence quantitative PCR experiment have been carefully selected and optimized based on our years of experimental experience. Most of them are imported products, with priority given to quality and then cost.
4. With years of experience in fluorescence quantitative PCR experiments, the batch processing and assembly-line operation method reduces the probability of individual experimental errors and variations.
Delivery content
1. RNA Extraction and Concentration Table
2. Detection data (default: three repetitions), 2-ΔΔCt analysis data
3. Relative quantitative comparison bar chart
4. Amplification curve and melting curve graph
5. Fluorescence Quantitative PCR Test Report
Material sample processing and preservation
1. Sampling method and precautions
- Quickly collect.
- The samples need to be collected, prepared, stored and transported as quickly and promptly as possible. The period from sample collection to experimentation should be shortened as much as possible to ensure the quality and stability of the samples.
- Low-temperature preservation (fresh sample samples) .
- After collecting the samples, if there are any separation steps that require to be carried out under low-temperature conditions such as 4℃ or on ice, the separated samples should be immediately placed in liquid nitrogen, dry ice or a -80℃ refrigerator, and ensure that they are below -80℃ before the experiment.
2. Sample submission of materials
Type Sample Submission Standards Conditions of carriage RNA sample
Volume > 20 μL, high-quality and non-degraded RNA is required. A gel picture for agarose electrophoresis should be provided, showing clearly two bands of 28S and 18S. The amount of dry ice required is 5 kg for 1 day, 10-20 kg for 2-3 days, and 20-30 kg for 3-5 days. It should be sealed. Material sample The weight of a single sample > 1g. Case Show
Figure: Calculation results of real-time fluorescent quantitative PCR detection
Figure: Amplification Curve Chart
Figure: Melting Curve Peak Graph
Q&A
1. Why is it necessary to limit the amplified fragment to a range of 80-300 base pairs?
Due to the presence of primer dimers, when the amplification sequence is too short, it is prone to be indistinguishable from the primer dimers; when the amplification sequence is too long, it will lead to a decrease in amplification efficiency and a decline in the accuracy of the detection results.
2. Relative quantification versus absolute quantification?
Absolute quantification refers to directly calculating the initial copy number of the sample to be tested through qPCR.
Relative quantification refers to the change in the amount of the target gene in the specified sample compared to that of another control sample.
3. Why is it necessary to provide a control group when an internal reference is also provided?
Many people may have such a question: Why do we need to confirm the reference gene when setting up a control group? In QPCR, the reference gene needs to be added for calibration and correction, to eliminate the possible differences in RNA yield, quality and reverse transcription among different samples, so as to obtain the true differences in the specific expression of the target gene. The control group is used for the calculation of relative expression levels in the analysis of experimental results.
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Tel: +86 025-85205672
Email: info@pronetbio.com
Address: Building 3C, Nanjing Xianlin Zhigu,
Qixia District, Nanjing, Jiangsu, China, 210033
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