A method for gene quantitative analysis

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...

AI Technical Summary

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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