Pyrimidines antimetabolic chemotherapeutical medicine curative effect prognose reagent kit and application thereof

A chemotherapeutic drug and kit technology, applied in the fields of biotechnology and clinical medical testing, can solve problems such as poor prognosis

Inactive Publication Date: 2007-06-06
GUANGDONG GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many meaningful phenomena have also been observed in traditional chemotherapy. Patients with low expression of ERCC1 have better response to platinum drugs, and those with low expression of β-tubulin III have better response to taxane drugs. The sensitivity of gemcitabine therapy is related to the expression level of nucleotide reductase M1 subunit in tumor tissue, and the prognosis of patients with high RRM1 expression is poor when receiving gemcitabine therapy

Method used

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  • Pyrimidines antimetabolic chemotherapeutical medicine curative effect prognose reagent kit and application thereof
  • Pyrimidines antimetabolic chemotherapeutical medicine curative effect prognose reagent kit and application thereof
  • Pyrimidines antimetabolic chemotherapeutical medicine curative effect prognose reagent kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Extraction of tissue RNA

[0026] 1. After the fresh tissue is removed, it is immediately placed in liquid nitrogen, and then stored in a -80°C refrigerator for detection.

[0027] 2. Take an appropriate size tissue specimen (about 100 mg) and grind it with platinum in liquid nitrogen, transfer it to a pre-cooled 1.5 ml centrifuge tube containing 1000 ul Trizol, and place it on ice for 10 minutes.

[0028] 3. Add 200ul of chloroform.

[0029] 4. Use tweezers to take the bullet, cover it, and mix it upside down for about 15 seconds.

[0030] 5. Let stand for 5 minutes.

[0031] 6. Centrifuge, 12000RPM / min, 4°C, 15min.

[0032] 7. Take the upper layer. (Middle and lower layer is a mixture of protein and DNA)

[0033] 8. Add isopropanol: Add 500ul isopropanol to 1000ml Trizol.

[0034] 9. Mix well and let stand for 10min. Centrifuge, 12000RPM / min, 4°C, 15min.

[0035] 10. Discard the supernatant directly, and you can see a little white precipitate sticki...

Embodiment 2

[0043] Embodiment 2: RT-PCR synthetic cDNA

[0044] 1. Mix all the reagents in the kit and collect by rapid centrifugation;

[0045] 2. Add the following reactants to a 0.2ml PCR reaction tube:

[0046] 1ug total RNA nul

[0047] 50uM oligo(dT)20 1ul

[0048] 10mM dNTP mix 1ul

[0049] DEPC-treatedwater made up to 10ul

[0050] Incubate at 3.65°C for 5 minutes and immediately place on ice for at least 1 minute.

[0051] 4. Add the following reactants to this tube and mix well.

[0052] 10XRT buffer 2ul

[0053] 25mM MgCl2 4ul

[0054] 0.1M DTT 2ul

[0055] RNASEOUT™ (40U / ul) 1ul

[0056] SuperScript™ III RT (200U / ul) 1ul

[0057] Incubate at 50°C for 50 minutes, then react at 85°C for 5 minutes. Place on ice immediately.

[0058] 6. Collect the product by centrifugation. Add 1ul RNase H and incubate at 37°C for 20 minutes.

[0059] 7. After the cDNA is synthesized, store it at -20°C for downstream work.

Embodiment 3

[0060] Embodiment 3: the construction of standard product

[0061] 1. Centrifuge PGEM-T Vector quickly, and collect the reaction product in a tube.

[0062] 2. Establish the connection system: put the following reactants in a 0.2mlL centrifuge tube:

[0063] 2X Rapid Ligation Buffer 5ul

[0064] pGEM-T Vector 1ul

[0065] PCR Product 2ul

[0066] T4DNA Ligase (3U / ul) 1ul

[0067] Sterile water 1ul

[0068] 3. Mix well and incubate at room temperature for 1 hour or overnight at 4°C.

[0069] 4. Prepare two AMP LB plates and store them at room temperature for later use.

[0070] 5. Take 2ul of the reactant in the connection system and put it into a 7ml centrifuge tube.

[0071] 6. Take 50ul of competent cells in a 7ml tube, flick and mix well, and ice-bath for 30 minutes.

[0072] Heat shock in a water bath at 7.42°C for 90 seconds. Immediately place on ice for 2 min.

[0073] 8. Add 950ul soc culture-based tube. Incubate at 37°C, 150rpm for 1.5 hours.

[0074] 9. Ce...

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PUM

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Abstract

The invention discloses a regent box to detect the expressing level in the tumor organize of the ribonucleotide reductase M1, RRM1. It can detect the malignancy sufferer who can resist on the metabolizing medicine curing.

Description

[technical field] [0001] The invention relates to a kit for detecting the expression level of human nucleotide reductase M1 subunit (ribonucleotide reductase M1, RRM1) in tumor tissues, so as to predict the curative effect of gemcitabine, an antimetabolite chemotherapeutic drug of pyrimidines, clinically. Good patient. Belongs to the field of biotechnology and clinical medicine testing. [Background technique] [0002] Lung cancer has become the tumor with the highest incidence and mortality worldwide due to air pollution, smoking and other factors brought about by the improvement of the global industrialization level. In my country, the incidence of lung cancer has increased by 11% annually in the past two decades, and it is estimated that by 2025, there will be more than 1 million new lung cancer patients in my country every year. According to the pathological classification, about 80% of the diagnosed lung cancers are non-small cell lung cancer (non-small-cell lung cancer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N21/17C12Q1/68
Inventor 吴一龙董嵩郭爱林陈志红张绪超林嘉颖谢至陈世良聂强
Owner GUANGDONG GENERAL HOSPITAL
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