Fluorescence quantitative PCR experimental method design |
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When designing a fluorescence quantitative experiment, how should I choose the quantification method? Relative or absolute quantification? Relative standard curve method or ΔΔCT method? The fluorescence quantitative PCR experimental method should be designed according to the experimental purpose. Generally speaking, there are two purposes to be achieved by fluorescence quantitative PCR: 1. Detecting the exact amount of a gene or species in a sample is called absolute quantification. For example, the amount of a virus in a certain volume of blood, or the amount of a certain bacteria in a certain body of water. 2. Detecting the relative expression of a gene in different samples is called relative quantification. For example, after different treatments, the expression of a gene in plant tissues is up-regulated or down-regulated. |
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Preliminary experiments are important Before we elaborate on the specific experimental design of absolute quantification and relative quantification, let us first understand what is the prerequisite for accurate quantification of samples in fluorescence quantification experiments? High accuracy - the fluorescence signal-CT value is in a standard linear relationship with the target product; Good repeatability - 3 or more repeated operations with a small error range; Wide dynamic range - accurate detection over a wide concentration range. We can judge the above requirements through preliminary experiments: Based on the introduction of primer design in our previous article, we can design specific quantitative primers and use them to design preliminary experiments: Primer and operation verification Perform a series of gradient dilutions on the nucleic acid of a sample (10-fold dilution at 5 points is recommended. If the cDNA concentration is too low, the dilution factor can be reduced). Set 3 or more replicates for each point and draw a standard curve. 2 ≥0.99, the experimental operation meets the requirements, the amplification efficiency E=90%-110%, and the primer amplification efficiency meets the requirements. |
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System detection Set up positive and negative controls. A positive control is a control group that can amplify 100% of the fluorescent signal, such as a standard or internal control. If the positive control does not amplify, it is necessary to check for system problems such as instruments and reagents. The negative control is a control group that cannot amplify fluorescent signals under normal circumstances. No template control (NTC): Use water as the template and observe the amplification. If amplification occurs with the NTC, the TM value of the melting curve can be used to preliminarily determine whether it is a product caused by contamination or primer dimers. No Reverse Transcription Control (NRC): Used in cDNA quantification experiments to monitor genomic contamination (this issue can also be avoided if quantitative primers can be designed across introns). During reverse transcription, a system is prepared without reverse transcriptase. All other components are added as normal, and the reverse transcription process is repeated. This is used as a template to monitor amplification. If genomic DNA is used as a template, only the NRC test is required. After the primers, operation, and system are verified, we can conduct quantitative experiments on the samples. In quantitative experiments, it is also necessary to set up replicates and controls. |
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Absolute quantification method Absolute quantification first requires the construction of a standard sample of known concentration. The standard sample is then used to construct a standard curve that plots the relationship between concentration and CT value, thereby calculating the concentration of the sample. Acquisition of standards Use quantitative primers to amplify the target product, clone and extract the plasmid, and use the plasmid as the standard; use specific primers to amplify a product containing the target fragment, which is longer than the target fragment, and use this product as the standard; use in vitro transcribed RNA as the standard. Standard concentration determination The absolute concentration of the standard must be determined by an independent method. Standard concentrations can be measured spectrophotometrically at A260 (requiring high sample purity) or using a fluorescent dye assay (Qubit, microplate reader, etc.). Copy number concentrations are calculated based on mass concentration. Example: DNA standard concentration is 10ng/µL, standard is 2500bp, the formula is as follows: (6.02 x 10 twenty three ) x ( 10ng/µL x 10 -9 )/( 2500bp x 660)= 3.65 x 10 9 copies/µL Standard curve drawing According to the concentration of the standard, dilute to the appropriate concentration range and then perform continuous gradient dilution (5 points of 10-fold dilution are recommended). Sample concentration calculation Perform fluorescence quantitative reaction simultaneously with the standard. After the copy number concentration of the standard is set on the quantitative instrument, the copy number concentration of the sample will be automatically given according to the CT value of the sample, or it can be manually substituted into the standard curve formula to calculate the copy number concentration of the sample. |
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Relative quantification method Relative quantification, unlike absolute quantification, detects differences in expression levels. Therefore, to identify true differences, it is necessary to eliminate human-induced errors, such as errors in sample quality or volume, errors in sample extraction yield, and errors in reverse transcription. This is where the concept of an internal reference gene comes in. The purpose of an internal reference gene is to eliminate potential differences in RNA yield, quality, and reverse transcription efficiency between samples, thereby identifying true differences in the specific expression of the target gene. Select internal reference genes The selection criteria for internal reference genes are as follows: High or moderate expression, excluding those that are too high or too low; The expression level is not affected by any exogenous factors; No pseudogenes exist. Currently, commonly used internal reference genes include GAPDH, β-actin, 18S rRNA, etc. The expression of the same internal reference gene is not constant in different tissues, such as heart, brain, lung, liver, skeletal muscle, and kidney. |
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Relative quantification method Relative quantification, unlike absolute quantification, detects differences in expression levels. Therefore, to identify true differences, it is necessary to eliminate human-induced errors, such as errors in sample quality or volume, errors in sample extraction yield, and errors in reverse transcription. This is where the concept of an internal reference gene comes in. The purpose of an internal reference gene is to eliminate potential differences in RNA yield, quality, and reverse transcription efficiency between samples, thereby identifying true differences in the specific expression of the target gene. Select internal reference genes The selection criteria for internal reference genes are as follows: High or moderate expression, excluding those that are too high or too low; The expression level is not affected by any exogenous factors; No pseudogenes exist. Currently, commonly used internal reference genes include GAPDH, β-actin, 18S rRNA, etc. The expression of the same internal reference gene is not constant in different tissues, such as heart, brain, lung, liver, skeletal muscle, and kidney. |
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The expression of the same internal reference gene may vary in different states within the same tissue. For example, the GAPDH gene is a key gene in glycolysis. During cell proliferation, glycolysis increases, and GAPDH expression is likely to be upregulated. If GAPDH expression is inconsistent when comparing tumor tissue with normal cells and adjacent normal tissue, careful selection of the internal reference is recommended. Due to the non-constancy of internal reference genes, qPCR experiments for internal reference gene selection (sampling quality or volume control, nucleic acid concentration control, etc.) are added during experimental design. The expression stability of internal reference genes in experimental samples is tested experimentally to select the most stable internal reference gene; alternatively, two or three internal reference genes are selected and the average value is calculated as the internal reference data. After selecting the internal reference gene, we can conduct relative quantification experiments. The relative quantification method is divided into the standard curve method and the ΔΔCT method. Relative standard curve method Relative standard curve method experimental design: Use the target gene and reference gene standards to generate standard curves. (The standard curve here does not require the exact concentration of the standard, so you can also use cDNA as the standard for serial dilutions, and use the multiple relationship as the standard concentration.) Amplify the target gene and the internal reference gene simultaneously in the treated and control samples to obtain the CT value, and use the internal reference gene to normalize the target gene; After normalization, the expression difference of the target gene in the treated sample and the control sample is obtained. Characteristics of relative standard curve method: Taking into account the differences in amplification efficiency of different genes, a standard curve is used to correct the amplification efficiency, thus minimizing errors; The amplification efficiency requirement for primers is not as high as that of the ΔΔCT method; The disadvantage is that a standard curve must be prepared for the target gene and the internal reference gene in each experiment; This method is suitable for experiments with large sample sizes but a small number of target genes to be analyzed. |
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ΔΔCT method Experimental design of ΔΔCT method: The premise for using the ΔΔCT method is that the amplification efficiency of the target gene and the internal reference gene are close to 100%, and the deviation is within 5%; Subtract the CT value of the reference gene from the CT value of the target gene to obtain the ΔCT value of the sample. Subtract the ΔCT of the control sample from the ΔCT of the treated sample to obtain ΔΔCT. Use Formula 2 - ΔΔCT The expression differences between treated samples and control samples were calculated. This method has a high throughput (no standard sample occupies the well position), requires high primer amplification efficiency, and is slightly less accurate than the relative standard curve method. |
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Throughout the experimental process, both biological and technical replications need to be considered. We will explain the experimental data analysis of specific quantitative results in subsequent corresponding topics. |
