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Method for comparing expression amount of idential gene in different origin using base sequence determination method

A technology of base sequence and expression, which is applied in the field of quantitative comparison of the relative expression of the same gene in tissues or cells of different origins, and achieves the effects of easy instrumentation, convenient operation and low price.

Inactive Publication Date: 2007-01-10
周国华
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since only 10-30 base sequences can be determined, this method is limited to the analysis of gene mutations and gene polymorphisms[9]

Method used

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  • Method for comparing expression amount of idential gene in different origin using base sequence determination method
  • Method for comparing expression amount of idential gene in different origin using base sequence determination method
  • Method for comparing expression amount of idential gene in different origin using base sequence determination method

Examples

Experimental program
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Effect test

Embodiment 1

[0024] [Example 1] Determination of the difference in the expression of P53 gene in human normal tissue, brain cancer tissue and liver cancer tissue.

[0025] This example describes the relative expression of P53 gene in three different source tissues by DNA adapter labeling method. Firstly, three different DNA adapters were designed, respectively connected with cDNA fragments digested by restriction endonucleases, and then mixed for PCR amplification.

[0026] 1. Preparation of cDNA samples

[0027](1) Extraction of total RNA: Take 0.1 g each of human normal tissue, brain cancer tissue and liver cancer tissue, add 1 ml Trizol to grind in a tissue grinder, and extract total RNA according to the instructions of Trizol. After the 28s and 18s bands were identified by electrophoresis without degradation, the concentration was determined by ultraviolet absorption spectrometry, and then sterilized DEPC-H 2 O adjusted the final concentration to 1 μg / μl.

[0028] (2) Synthesis of t...

Embodiment 2

[0041] [Example 2] Determination of the difference in the expression of P53 gene between human liver cancer cells and bladder cancer cells.

[0042] This example mainly uses the reverse transcription primer labeling method to determine the difference in the expression of the P53 gene in human liver cancer cells and bladder cancer cells, that is, primers with different sequences are used to reverse transcribe mRNA from different sources, so that the cDNA from each source is labeled DNA fragments with different sequences. And compared with the results of RT-PCR.

[0043] 1. Preparation of Assay Samples

[0044] According to the method of [Example 1], total RNA was extracted from human liver cancer cells and bladder cancer cells respectively. After the quality was verified by electrophoresis, the concentration was measured by ultraviolet light, and then the final concentration was adjusted to 1 μg / μl with DEPC-H20. The mRNA in human liver cancer cells and bladder cancer cells w...

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Abstract

The present invention relates to an analytical method for quantitatively comparing expression amount of identical gene in different origins, wherein the gene expression amount difference of the identical gene in different source tissues or cells are quantitatively compared, by adopting the quantitatively specific characters of bioluminescence method and the principle that different deoxyribonucleoside triphosphates (dNTP) are added therein one by one. The concrete steps are as follows: the messenger RNAs (mRNA) from different sources are inverse transcribing into cDNA, and a section source specificity DNA sequence for cDNA from each source is signed; the signed cDNA from different sources are mixed in a tube, as a substrate of a polyase chain reaction (PCR); a common primer and a gene specificity primer are used to execute PCR enlargement; bioluminescence analytical method is adopted to determine the base sequences, wherein the base species represent different sources of genes, and the signal intensity represents the expression amount of genes form each source. Said method exhibits great significance for disease related gene selecting, clinical early diagnosis and creation of specific medicines for treating diseases.

Description

technical field [0001] The present invention relates to a method for quantitatively comparing the relative expression of the same gene in tissues or cells of different origins, specifically a method for simultaneously measuring DNA fragments in a mixture of DNA fragments marked with different base sequences by using base sequencing method of relative content. Background technique [0002] With the advancement of molecular biology and analytical instruments, the sequencing work of the Human Genome Project has been completed, and now it has shifted from structural analysis to functional analysis [1], that is, to understand the distribution of gene transcription product mRNA and the translation product protein of mRNA in cells. And the existence, distribution and function in organs and tissues. By comparing gene expression levels between healthy and diseased individuals, disease-related genes can be searched for and discovered, and then used for early clinical diagnosis [2]. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 周国华
Owner 周国华
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