Detection method of miRNA absolute expression level in biological sample

Abstract

The invention relates a method for measuring miRNA absolute expression quantity in biological samples, which comprises the following steps: 1) material selection: 8 weeks and 40 weeks of mouse brain tissues are selected; 2) total RNA extract; 3) RT(reverse transcription)-Polymerase Chain Reaction (PCR); 4) quantifying DNA; 5) drawing specification curve; 6) data processing: loss rate and average loss rate of little RNA is figured out in the process of extracting RNA according to the copy number of spike RNA in the extracted total RNA which is detected by the PCR; the expression quantity of every initial cell of the little RNA at the levels of DNA and RNA is figured out according to the cell number of original sample that is respectively expressed from the levels of DNA and RNA. The method has the advantages of miRNA expression quantity is faithfully transformed into the absolute expression quantity in the original sample through the cell number in the sample homogenate which is worked out from the two levels of DNA and RNA; the disadvantage that relative differentiation can only be reflected by taking housekeeping genes as an internal reference, and the analysis is truer and more reliable from the two levels of DNA and RNA.

Description

technical field [0001] The invention relates to a method for detecting biological samples in the field of biotechnology, mainly a method for detecting the absolute expression of miRNA in biological samples, which is suitable for detecting the absolute expression of other target genes in single cells from biological samples. Background technique [0002] Since Lee first discovered miRNAs with timing regulation in C. elegans in 1993, the expression regulation of miRNAs has been widely recognized. With the rapid development of molecular biology technology, especially the continuous update of miRNA detection technology, such as cloning technology, chip technology and real-time fluorescent quantitative PCR (polymerase chain reaction) technology, more and more miRNAs have been discovered. . At present, the quantitative detection and functional research of miRNA has become a hot topic in this field. However, to faithfully restore the original expression levels of miRNAs from the ...

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Problems solved by technology

However, this normalization method can only reflect the changes in the expression of target genes in different periods, but cannot reflect the absolute copy number of changes.

[0004] miRNA is a kind of small molecule RNA with temporal expression regulation function. Its expression level will change in different stages of organism development and maturation according to the needs of regulation, while "housekeeping genes" are different in different growth stages or under different environmental stresses. Afterwards, its expression level will also change, which determines that the traditional homogenization method using the housekeeping gene as an internal reference can only reflect the relative differences of the target gene in different periods, but cannot faithfully reflect the absolute expression level of the target gene in each period

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