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mRNA difference displaying method with ordered robustness

A differential display and robust technology, applied in the field of robust and orderly differential display of mRNA, can solve the problems of dependence and little effect, and achieve the effect of credible results, high coverage and economical operation cost

Inactive Publication Date: 2012-03-07
WUHAN UNIV
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, coverage is a tricky issue for it, which depends on the distribution of the two kinds of restriction sites, especially the low-frequency restriction sites, in each cDNA
More than a dozen methods have been tried to overcome this problem with little success

Method used

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  • mRNA difference displaying method with ordered robustness
  • mRNA difference displaying method with ordered robustness
  • mRNA difference displaying method with ordered robustness

Examples

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Embodiment 1

[0033] Example 1: Study on gene expression differences between leaves and roots of one-week-old rice seedlings:

[0034] A method for robust and ordered mRNA differential display, comprising the following steps:

[0035] (1) Synthesis of double-stranded cDNA. Total RNA was extracted from middle leaves and roots of one-week-old standard japonica rice Nipponbare seedlings whose genome sequence had been determined using TRIzol reagent (Invitrogen, USA). After DNase I (Promega, USA) treatment, phenolform (volume ratio 1:1) extraction and 75% (volume percentage) ethanol precipitation, the purified total RNA was dissolved in 30 μl diethyl pyrocarbonate (DEPC) treated of water. Take an appropriate amount of purified total RNA from leaves and roots, use the designed bmaT16V as a primer (synthesized by Invitrogen, USA), and use SuperScript TM III reverse transcriptase (Invitrogen, USA) was used to synthesize the first strand of cDNA respectively. Further use RNase H, DNA polymeras...

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Abstract

The invention discloses an mRNA difference displaying method with ordered robustness, comprising the following steps: A. extracting total RNA, utilizing poly(T) primer with a 5'-end biotin label and an Acul restriction enzyme cutting site for reverse transcription to synthesize a cDNA first link, and double links are further synthesized with a cut transition method; B. purifying and recycling cDNA, utilizing MspI for digestion, and utilizing T4 ligase to connect an MspI joint; C. utilizing a magnetic bead method to separate a fragment with biotin, and eluting residual fragments; D. utilizing AcuI for secondary digestion to the fragment with biotin, and utilizing the T4 ligase to connect an AcuI force joint; E. pre-amplifying normal cDNA-AFLP and performing selective pre-amplification; andF. displaying urea modification PAGE gel electrophoresis and differential amplification fragments, wherein, the differential amplification fragments can be cut off for recovery, re-expansion, clone and sequence measurement for further analysis. The invention has higher sensitivity, accuracy, coverage rate, high flux and repetitiveness and is suitable for normal molecular biology laboratories to carry out various differential expression researches.

Description

technical field [0001] The invention relates to the technical field of whole gene expression map of molecular biology and genetics. More specifically, it relates to a method of robust and ordered mRNA differential display, which is applicable to the comparative study of mRNA differences in the same tissue cells of all eukaryotes at different developmental stages and under different physiological and pathological conditions, or different tissue cells in the same Comparative studies of mRNA differences during developmental periods or under the same physiological and pathological conditions. Background technique [0002] Genome-wide expression mapping technology is one of the core technologies in the field of modern life science research. At present, the complete genome sequences of some important model eukaryotes such as yeast, Drosophila, nematode, human, mouse, Arabidopsis, rice and sorghum have been published, but the genomes of a large number of biological species are sti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 丁毅刘宏高
Owner WUHAN UNIV
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