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Oligonucleotide and method for detecting ABCG2 gene relative expression quantity in sample

A relative expression level, in-sample technology, applied in the field of detecting the relative expression level of the ABCG2 gene in the sample, can solve the problems of high cost and poor specificity, and achieve the effect of simple operation, high sensitivity, and improved experimental efficiency

Pending Publication Date: 2020-03-24
南京艾迪康医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, because SYBR GreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman method, and its specificity must be judged by observing the melting curve; and the cost of the double-probe hybridization method is relatively expensive.

Method used

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  • Oligonucleotide and method for detecting ABCG2 gene relative expression quantity in sample
  • Oligonucleotide and method for detecting ABCG2 gene relative expression quantity in sample
  • Oligonucleotide and method for detecting ABCG2 gene relative expression quantity in sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The nucleic acid detection method used to detect the relative expression level of ABCG2 in samples can be used to assist in the formulation of lung cancer chemotherapy regimens and the selection of chemotherapy drug doses in clinic, and can also predict the prognosis of patients. The method includes:

[0050] (1) Red blood cell lysate, including 16 μmol / L ammonium chloride, 1 mmol / L potassium bicarbonate and 12.5 μmol / L EDTA.

[0051] (2) RNA extraction reagents, including TRIzol, chloroform, isopropanol, 75% ethanol and RNase-free water.

[0052] (3) RNA reverse transcription reagent, ReverTra Ace qPCR RT Kit kit (TOYOBO company).

[0053] (4) Detection system PCR reaction solution, THNDERBIRD Probe qPCR Mix (2×) (TOYOBO Company). Detect the upstream primer ABCG2-F, downstream primer ABCG2-R and probe ABCG2-Probe of the ABCG2 gene; detect the upstream primer actin-F, downstream primer actin-R and probe actin-Probe of the internal reference gene actin. The primer and...

Embodiment 2

[0062] 1. The operation process of total RNA extraction from peripheral blood samples:

[0063] (1) Collect 1ml of anticoagulated fresh blood, and register age and gender;

[0064] (2) Add 1ml of erythrocyte lysate into a clean 1.5ml centrifuge tube, take 0.5ml of anticoagulated blood and mix well.

[0065] Stand at room temperature for 10 minutes;

[0066] (3) Centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom;

[0067] (4) Add 0.5ml red blood cell lysate again, centrifuge at 5000rpm for 5min, discard the supernatant, and collect the cells at the bottom;

[0068] (5) Add 1ml TRIzol to the cells, pipette repeatedly until the precipitate is completely dissolved, and let stand at room temperature for 5 minutes;

[0069] (6) Add 0.2ml chloroform and shake evenly;

[0070] (7) Centrifuge at 14000rpm at 4°C for 10min, absorb the supernatant layer and transfer it to another new centrifuge tube (do not absorb the white middle layer);

[...

Embodiment 3

[0087] The method of the invention is used to detect the peripheral blood samples of the healthy population.

[0088] Take 24 cases of peripheral blood samples from healthy people for examination, extract RNA according to the method described in Example 2, perform reverse transcription, prepare reagents and perform fluorescence quantitative PCR detection.

[0089] For each sample, 2 μL of cDNA obtained by reverse transcription was added to the detection system PCR reaction solution. At the same time, make one copy of positive control, negative control and blank control, and two copies of standard curve of internal reference gene / target gene. A 96-well fluorescent PCR instrument can detect 16 samples at the same time, each sample is repeated twice, a positive control, a negative control and a blank control. The detection time is only 100 minutes.

[0090] Test results such as Figure 5 As mentioned above, the normal human blood cDNA was used as a template to detect ABCG2 and...

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Abstract

The invention provides oligonucleotide and a method for detecting ABCG2 gene relative expression quantity in a sample. According to a double-standard-curve method, quantitative standard curves of a reference gene actin and an ABCG2 target gene are constructed respectively by combining a real-time fluorescence PCR technology with a Tapman probe; and expression of ABCG2 in the body of a testee is detected. According to the invention, accurate detection is realized; the test sensitivity is high; and the operation is simple and convenient. A reference and basis can be provided for formulation of chemotherapy regimens of lung cancer patients and individualized treatment of the patients is facilitated.

Description

technical field [0001] The invention belongs to the fields of biological science and biotechnology, and in particular relates to a method for detecting the relative expression level of ABCG2 gene in a sample. Fluorescence quantitative PCR technology is used to detect the expression level of ABCG2 gene in tumor patients. Background technique [0002] Adenosine triphosphate-binding transporter G2 (ATP-Binding Cassette Family G member 2, ABCG2), also known as breast cancer resistant protein (BCRP), is one of the members of the ABC transporter superfamily and is an important transmembrane transporter. The ABCG2 gene is located on human chromosome 4q22 and encodes 655 amino acids. These proteins all have ATP-binding sites, which provide energy by hydrolyzing ATP to transport substrates against the cell membrane or into organelles. The transport substrates include various drugs / toxins and their metabolites, physiological metabolites, nutrients, Lipids and peptides etc. A large ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/106C12Q2600/118C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/114C12Q2561/101
Inventor 牛林梅李允章王淑一
Owner 南京艾迪康医学检验所有限公司
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