Biological chip for detecting control of related gene expression by pharmaceutical active component

A gene expression and biochip technology, which is applied in the field of biochips to detect the regulation of drug active ingredients on the expression of related genes, can solve the problems of numerous genes, unsuitable applications, and high costs

Inactive Publication Date: 2008-03-05
LOGISTICS UNIV OF CAPF
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these constructed gene chips involve a large number of genes, and the cost is very high, so they are not suitable for application in the research of a certain project (antitumor drugs, etc.)

Method used

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  • Biological chip for detecting control of related gene expression by pharmaceutical active component

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] drug treated cells

[0082] 1. Cell culture

[0083] K562 cells were cultured in RPMI1640 (pH 7.0) medium containing 10% fetal bovine serum and 80 U / L gentamicin at 37°C and 5% CO 2 Growth was incubated in a constant temperature incubator.

[0084] 2. Treatment of cells with antineoplastic drug artemisinin

[0085] Weigh a certain amount of penicillin and dissolve it in 500 μl DMSO, and heat to help dissolve it. Then diluted with serum-free medium to a concentration of 256×10 -5 mol / L. Before use, dilute to the required concentration with serum-free medium, respectively 1×10 -5 mol / L, 4×10 -5 mol / L, 16×10 -5 mol / L, 64×10 -5 mol / L, 256×10 -5 mol / L. Routinely cultured cells, adjusting the number of cells to 1×10 5 Each / mL, add 0.5ml of the above-mentioned drugs of each concentration. to a final concentration of 1 x 10 -6 mol / L, 4×10 -6 mol / L, 16×10 -6 mol / L, 64×10 -6 mol / L, 256×10 -6 mol / L. The cells were then placed at 37°C, 5% CO 2 Incubate in the inc...

Embodiment 2

[0087] TRIzol reagent for RNA extraction

[0088] Collect the cells into a centrifuge tube after 24 hours of drug action, discard the culture medium, invert to air dry, add 1ml TRIzol reagent, pipette repeatedly, transfer to a 1.5ml Eppendorf tube, and place at 15-30°C for 5 minutes. Add 0.2ml of chloroform (trichloromethane), vibrate vigorously for 15 seconds; then continue to stand at 15-30°C for 2-3 minutes; centrifuge at 12000×g for 15 minutes at 2-8°C. After centrifugation, take the upper colorless RNA-containing aqueous phase (about 360ul) to a new 1.5ml Eppendorf tube. Do not suck the solution below the water phase, so as not to contaminate DNA and protein. Add 0.5ml of isopropanol, let stand at 15-30°C for 10 minutes, then centrifuge at 12000×g for 10 minutes at 2-8°C. Discard the supernatant, add at least 1ml of pre-cooled 75% ethanol to wash the RNA pellet once. Shake vigorously to mix the sample and centrifuge at 2-8°C at least 7500×g for 5 minutes. Discard the ...

Embodiment 3

[0090] cDNA generation - reverse transcription direct labeling experiments

[0091]Preparation system 1: 10-40 μg total RNA, ≤11 μl; Anchored Oligo (dT) 20 Primer (2.5 μg / μl), 2 μl, RNase-Free water to 13 μl, mix well, briefly centrifuge for 5 seconds, heat at 70°C for 10 minutes, then quickly Place on ice and incubate for at least 1 min. Configuration System 2: System 1, 13μl; 5X First-Strand buffer, 6μl; (0.1M) DTT3μl, dNTP Mix for labeled, 2μl; RNaseOUT TM (40U / μl), 1μl; Labeled dCTP (1mM) 3μl; SuperScript TM III RT (400U / μl), 2μl; total volume 30μl, mix well, briefly centrifuge for 5 seconds, incubate at 46°C for 3 hours, and protect from light during the incubation period. After the incubation, add 15ul of 0.1M NaOH, mix well, and heat at 70°C for 30 minutes. Add 15ul 0.1M HCl and mix well.

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Abstract

The present invention discloses one kind of biochip for detecting the regulation and control of active medicine components on the expression of relevant genes. On one glass chip, one silicon chip or polymer chip, are fixed gene DNA probes for detecting PCNA, CyclinA1, CyclinB1, CyclinC, CyclinD1, CyclinD1, CyclinE1, CyclinG1, Cdk4, Cdk2, Cdc2, p16, p21, p53, E2F1, Chk1, Chk2, ATM, DNA-PK, DNA-Topo I, hTERT, Bcl-2, Trail, Mcl-1, Bax, Bak1, Brca1, Caspase3, Erk-3, Jnk, p38, Cdc25A, Fgd1, Rac and VEGF. The present invention can detect the regulation and control of active medicine components on the expression of relevant genes, and has the features of real-time, high sensitivity, high accuracy, high efficiency, etc.

Description

technical field [0001] The invention relates to a biological chip, in particular to a biological chip for detecting the regulation of the active ingredients of medicines on the expression of related genes. Background technique [0002] Modern pharmacological research has made it clear that the drug action has its target gene, and the target gene is the most essential indicator of the drug action. Therefore, in drug research, it is very important to identify genes with different expression before and after drug action, which plays a vital role in the development and application of drugs. With the development of related technologies in molecular biology, more and more technologies have been used to analyze and isolate differentially expressed genes. At present, the methods for large-scale screening of differentially expressed genes mainly include differential display RT-PCR (DDRT-PCR), subtractive hybridization (SH), and systematic analysis of gene expression (serial analysis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 陈立军姚丽呼文亮
Owner LOGISTICS UNIV OF CAPF
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