Biologically phenotypic RNA array of high-flux detection of mRNA expression abundance

A messenger RNA, RNA technology, applied in the field of biological phenotype RNA array, can solve problems such as undeveloped technology

Inactive Publication Date: 2003-01-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although cDNA microchip technology has been widely used, the technology of biological phenotypic ribonucleic acid array prepared by using RNA as sample preparation has not been developed.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1. Extraction of total RNA

[0050] Total RNA was prepared using the method of Sambrook et al., (1989).

[0051] The rice endosperm was collected and stored at -20°C for later use. Take out 100mg of tissue, add 1ml solution D (containing 4M guanidine isothiocyanate, 25mM sodium citrate (pH7.0), 5% sodium lauryl sarcosine (Sarcosyl), 0.1M β-mercaptoethanol), and the tissue Fully homogenate, transfer the homogenate into a 10ml centrifuge tube, then add 0.1ml of 2M sodium acetate (pH4.0), 1ml of water-saturated phenol and 0.2ml of chloroform / isoamyl alcohol (49:1) mixture, fully After shaking and mixing, let stand on ice for 15 minutes. The sample was centrifuged (10000g) at 4°C for 20 minutes, the upper aqueous phase was absorbed, mixed with an equal volume of isopropanol, and frozen at -20°C for at least 1 hour. Centrifuge at 4°C (10000g) for 20 minutes to collect the precipitate and dissolve it in 0.3ml solution D, add 0.03ml 2M sodium acetate and 0.3ml isopropanol, ...

Embodiment 2

[0053] Plant Genomic DNA Extraction

[0054] Add 20ml of extraction buffer I to a 50ml centrifuge tube and preheat in a 60°C water bath. Rice seedlings or leaves 5-10g, cut into pieces, add liquid nitrogen in a mortar, grind into powder, immediately pour into a preheated centrifuge tube, shake vigorously to mix, keep warm in a 60°C water bath for 30-60min (long time, DNA high yield), shaking occasionally. Add 20ml of chloroform / amyl alcohol / ethanol solution, mix by inversion (gloves are required to prevent damage to the skin), let stand at room temperature for 5-10 minutes, and separate the aqueous phase and the organic phase (re-mix if necessary). Centrifuge at 5000rpm for 5min at room temperature. Carefully pipette the supernatant to another 50ml centrifuge tube, add 1 times the volume of isopropanol, mix well, and leave it at room temperature for a while to form flocculent DNA precipitates. Add 1ml TE to 1.5ml eppendorf. Use a hooked glass rod to remove the DNA flocs, b...

Embodiment 3

[0056] Extraction of animal genomic DNA

[0057] Take about 5g of tissue, remove the connective tissue, absorb the blood with absorbent paper, cut it into pieces and put it in a mortar (the finer the better). Pour into liquid nitrogen, grind into powder, add 10ml separation buffer. Add 1ml of 10% SDS, mix well, and the sample becomes very viscous at this time. Add 50 μl or 1 mg of proteinase K, and incubate at 37°C for 1 to 2 hours until the tissue is completely disintegrated. Add 1ml of 5mol / L NaCl, mix well, and centrifuge at 5000rpm for a few seconds. Take the supernatant in a new centrifuge tube and extract with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1). Centrifuge at 3000rpm for 5min after stratification. Take the upper aqueous phase to a clean centrifuge tube, add 2 times the volume of ether for extraction (operate under ventilated conditions). The upper ether was removed, and the lower aqueous phase was retained. Add 1 / 10 volume of 3mol / L NaAc...

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Abstract

A biologically phenotypic RNA array for high-flux detection of mRNA expression abundance features that different biological pheno typic RNAs are applied onto an array carrier, and each RNA has a particular concentration gradient, so said array has different concentration gradients for different samples, resulting in precisely quantitating particular mRNA. Said array can be used for disease diagnosis, gene detection and therapy, agricultural purpose, etc.

Description

technical field [0001] The technical field of the present invention is the field of biotechnology. Further, the present invention relates to a biological phenotype ribonucleic acid array for high-throughput detection of the expression abundance of messenger ribonucleic acid. Background technique [0002] The essence of a biochip (array) is to arrange a series of addressable biorecognition molecules fixed at a certain position in an orderly array on a small substrate (R.J.Lipshutz et al., Bio Techniques19 (1995), 442- 447; S.P.Fodor et al., Nature 364(1993), 555-556; S.P.Fodor et al., Science 251(1991), 767-773; A.C.Pease et al., Proc.Natl.Acad.Sci.USA 91 (1994), 5022-5026), by hybridizing with a labeled sample, detecting the strength of the hybridization signal and then judging the number of target molecules in the sample. This technology immobilizes a large number of nucleic acid sequence molecules on the carrier at the same time, detects and analyzes a larg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 董海涛李德葆
Owner ZHEJIANG UNIV
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