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A quantitative analysis and gene technology, applied in the fields of biotechnology and medicine, can solve problems such as unrealistic, affecting the clarity of large charts, and inconsistent meanings of CT
Active Publication Date: 2021-03-23
宜昌美光硅谷生命科技股份有限公司
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Problems solved by technology
When using the CT value directly for analysis and comparison, I encountered some problems. When the gene quantification results are shown in the chart, the larger the CT value, the lower the copy number of the gene at the beginning. Then, the undetectable gene is represented by "0" ", but it does not coincide with the meaning of CT; some software uses "999", which greatly affects the clarity of large charts
When doing gene quantitative analysis and comparison, it is also necessary to standardize the data of detection genes and housekeeping genes. At this time, such a large range of 999 brings a lot of inconvenience to the clarity of analysis results, especially in single-cell analysis. unrealistic
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Embodiment 1
[0029] A method for quantitative analysis of single-cell housekeeping gene beta-Actin, comprising the following steps:
[0030] 1. Take a group of samples containing one or a small number of cells, treat the cells with proteolytic enzyme (Proteinase K), use CellsDirect qRT-PCR kit (Invitrogen, USA) to synthesize cDNA, and pre-amplify the relevant fragments of beta-Actin gene 18cyces, used as beta-Actin gene quantification.
[0031] 2. With the gene template pre-amplified in step 1, use quantitative PCR equipment and reagents to detect the initial copy number of the beta-Actin gene in the pre-amplified sample, and obtain the original CT data, that is, the CT value, as shown in the table 1.
[0032] Table 1 Single-cell analysis of housekeeping gene beta-Actin
[0043] A method for quantitative analysis of the single-cell level of single-cell housekeeping genes GAPDH, CD45, EpCAM, CK7, CK8, CK18, and CK19 genes, comprising the following steps:
[0044] 1. Take a group of samples containing one or a small number of cells, treat the cells with proteolytic enzyme (Proteinase K), use CellsDirect qRT-PCR kit (Invitrogen, USA) to synthesize cDNA, and pre-amplify GAPDH, CD45, EpCAM, CK7 , CK8, CK18, and CK19 gene related fragments 18cyces, the gene template obtained by pre-amplification is used as the subsequent quantitative PCR;
[0045] 2. Use the gene template pre-amplified in step 1 for quantitative PCR test, and use quantitative PCR instruments and reagents to detect the initial copy number of multiple genes GAPDH, CD45, EpCAM, CK7, CK8, CK18, and CK19 in the same sample , to obtain the original CT data, that is, the CT value, as shown in Table 3.
[0046] Table 3 Raw CT values at the single-cell level of GAPDH, CD45, EpCAM, CK7, CK8...
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Abstract
The invention discloses a method for gene quantitative analysis. The method comprises the following steps: (1) preparing an initial sample, processing cells by virtue of a protein decomposition enzyme, and conducting related gene pre-amplification; (2) implementing a quantitative PCR test by virtue of an amplified gene template, and detecting the initial copy number of related genes, so as to obtain original CT data, namely a CT value; (3) in accordance with a quantitative PCR instrument and a reagent, setting the maximum reliable CT limit value, called maxCT value for short; (4) converting the obtained CT value into eCT in accordance with a computation formula: eCT=maxCT-CT; and (5) in accordance with the obtained eCT, conducting quantitative analysis and comparison on gene expression andgene copy number of single cells or a few of cells, so as to obtain single-cell related gene quantitative data which is applied to science and clinical diagnosis information such as cell functional analysis, qualitative distinction, source identification and the like. The method is simple and easy to implement and is direct; and detection results are more real and clearer and are more conducive to clear comparison, so that a better cell qualification and classification effect can be achieved.
Description
technical field [0001] The invention belongs to the fields of biotechnology and medical technology, and more specifically relates to a method for quantitative analysis of single-cell gene expression, which is suitable for the detection and quantification of single-cell gene expression, as the analysis and comparison of cell qualitative and cell tissue sources. Background technique [0002] When analyzing gene expression or gene quantification, the analysis and comparison methods of quantitative PCR and CT values are commonly used. However, the traditional CT value is inversely proportional to the gene amount, that is, the larger the CT value, the lower the actual gene amount, which brings a lot of inconvenience to the analysis and comparison results. Generally, when quantitative PCR instruments and related software are used to detect gene expression levels or related gene copy numbers, the first-hand information obtained is CT value (C stands for Cycle, T stands for Thresh...
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