Design of Fluorescence Quantitative PCR Experimental Method |
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When designing a fluorescence quantitative experiment, how should we choose the quantitative method? Relative quantification 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 for fluorescence quantitative PCR: 1. Detecting the exact content of a gene or species in a sample is called absolute quantification. For example, the content of a virus in a certain volume of blood, or the content 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 upregulated or downregulated. |
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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 has a standard linear relationship with the target product; Good repeatability - 3 or more repeated operations with small error range; Wide dynamic range - can detect accurately over a wide concentration range. We can judge the above requirements through preliminary experiments: According to the introduction of primer design in our previous article, we designed specific quantitative primers and used specific primers 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. By observing the standard curve, it is generally believed that R 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. The positive control is a control group that can amplify the fluorescent signal 100%, such as standard, internal reference, etc. If the positive control does not amplify, it is necessary to check the system problems such as instruments and reagents. The negative control is the control group that cannot amplify the fluorescent signal under normal circumstances. No template control (NTC): Use water as the template and observe the amplification. If amplification occurs in 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 quantitative experiments to monitor genomic contamination (if quantitative primers can be designed across introns, this problem can also be avoided). During reverse transcription, a system without reverse transcriptase is configured, and the other components are added normally. After the same reverse transcription process, this is used as a template to observe the amplification. If genomic DNA is used as a template, only NTC detection is required. After the primers, operation, system, etc. are verified, we can conduct quantitative experiments on samples. In quantitative experiments, it is also necessary to set up repetitions and controls. |
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Absolute quantification Absolute quantification first requires the construction of a standard sample with a known concentration, and the use of the standard sample to construct a standard curve that plots the correspondence 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 a standard; use specific primers to amplify a product containing the target fragment, which is longer than the target fragment, and use this product as a standard; use in vitro transcribed RNA as a standard. Determination of standard concentration The absolute concentration of the standard must be obtained by other independent methods. The concentration of the standard can be measured at A260 using a spectrophotometer (requires high sample purity), or the concentration can be determined using a fluorescent dye method (Qubit, microplate reader, etc.). The copy number concentration is calculated based on the 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 Relative quantification is different from absolute quantification in that it detects differences in expression levels. Therefore, in order to obtain the real differences, it is necessary to first eliminate errors introduced by humans. Such as sample sampling quality or volume errors, sample extraction yield errors, reverse transcription errors, etc. The concept of internal reference genes is introduced here. The role of internal reference genes is to remove the possible differences in RNA yield, quality, and reverse transcription efficiency between different samples, so as to obtain the real differences in the specific expression of the target gene. Select reference gene The selection criteria for internal reference genes are as follows: High or moderate expression, excluding too high or too low expression; 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, kidney, etc. |
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Relative quantification Relative quantification is different from absolute quantification in that it detects differences in expression levels. Therefore, in order to obtain the real differences, it is necessary to first eliminate errors introduced by humans. Such as sample sampling quality or volume errors, sample extraction yield errors, reverse transcription errors, etc. The concept of internal reference genes is introduced here. The role of internal reference genes is to remove the possible differences in RNA yield, quality, and reverse transcription efficiency between different samples, so as to obtain the real differences in the specific expression of the target gene. Select reference gene The selection criteria for internal reference genes are as follows: High or moderate expression, excluding too high or too low expression; 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, kidney, etc. |
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The same internal reference gene may have different expressions in different states of the same tissue. For example, the GAPDH gene is a key gene in glycolysis. During cell proliferation, glycolysis metabolism increases, and the expression level of the GAPDH gene is likely to be upregulated. For example, when comparing tumor tissue with normal cells and adjacent normal tissues, the expression of GAPDH is not consistent. In this case, it is recommended to carefully select the internal reference. Due to the non-constancy of the internal reference gene, qPCR experiments for the selection of the internal reference gene (sampling quality or volume control, nucleic acid concentration control, etc.) are added during the experimental design. The expression stability of the internal reference gene in the experimental sample is tested experimentally, and the most stable internal reference gene is selected; or 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 quantitative experiments. The relative quantitative 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 internal reference gene standards to obtain standard curves (the standard curve here does not require the exact concentration of the standard, so cDNA can also be used as a standard for continuous gradient dilution, and virtual numbers based on the multiple relationship are used as standard concentrations); · Amplify the target gene and the internal reference gene simultaneously in the treated and control samples, 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 was 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 avoiding errors to the greatest extent; The requirement for primer amplification efficiency is not as high as that of the ΔΔCT method; The disadvantage is that a standard curve must be made for the target gene and the internal reference gene in each experiment; This method is suitable for experiments with large sample size but a small number of target genes to be analyzed. |
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ΔΔCT method Experimental design of ΔΔCT method: The premise of 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 internal reference gene from the CT value of the target gene of the sample to obtain the ΔCT 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 difference in expression between the treated samples and the control samples was 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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During the entire experimental process, biological replication and technical replication need to be considered. We will explain the experimental data analysis of specific quantitative results in subsequent corresponding topics. |
