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Quantitative detection method and application of molecular marker SLC22A3

A technology for detecting substances and drugs, which is applied in the field of molecular biology and can solve problems such as meanings that need to be discovered urgently

Active Publication Date: 2021-06-04
ZHENJIANG NO 1 PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no protocol to detect the expression and methylation of SLC22A3 gene in AML, and the significance of SLC22A3 to leukemia still needs to be discovered urgently

Method used

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  • Quantitative detection method and application of molecular marker SLC22A3
  • Quantitative detection method and application of molecular marker SLC22A3
  • Quantitative detection method and application of molecular marker SLC22A3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Establishment of a method for detecting the expression level of SLC22A3 based on quantitative PCR

[0032] (1) Total RNA extraction:

[0033] ① Separation of bone marrow mononuclear cells: Add 5ml of lymphocyte separation medium and red blood cell lysate to the obtained bone marrow specimen successively, mix upside down, centrifuge at 1000×rpm for 5min, and discard the supernatant;

[0034] ② Add 1ml TRIZOL to the above precipitate, and fully pipette and mix until the water sample;

[0035] ③Add 200μl chloroform, vortex fully shake for 30sec, let stand at room temperature for 5min, and centrifuge at 12000×rpm for 10min;

[0036] ④Add the supernatant to a clean 1.5ml EP tube, add 600μl isopropanol at the same time, close the lid tightly, mix up and down 50 times, let stand at room temperature for 10min, centrifuge at 12000×rpm for 10min, carefully discard the supernatant, and transfer to the EP tube The bottom white precipitate is RNA;

[0037] ⑤ Add 1ml of...

Embodiment 2

[0052] Example 2: Design and synthesis of primers for specifically amplifying the CpG island of the SLC22A3 gene promoter region

[0053] Aiming at the CpG island information in the promoter region of SLC22A3 gene, two sets of primers were designed for methylated and unmethylated sequences. These two pairs of primers can be used to specifically amplify the methylated and unmethylated sequences of the CpG island in the promoter region of SLC22A3 respectively. Methylation sequence, followed by quantitative analysis of methylation, the specific design steps are as follows:

[0054] The SLC22A3 promoter CpG island sequence was found and exported at UCSC (http: / / genome.ucsc.edu / ):

[0055] Path: UCSC homepage→Tools→gene sorter→Genome(Human)→search(SLC22A3)→go→select SLC22A3→Genomic Sequence→select the 5’UTR of 2000bp upstream and 60bp downstream of the promoter→export the obtained sequence.

[0056] Use the Methyl primer 1.0 software to design the required MSP primers:

[0057] I...

Embodiment 3

[0060] Example 3: Establishment of a method for detecting the methylation status of the SLC22A3 gene promoter GpG island based on quantitative PCR

[0061] The detection method of the present invention is based on the principle of methylation-specific PCR, and methylated and non-methylated DNA sequences can be distinguished through quantitative PCR amplification containing methylated and unmethylated specific primers respectively . The method comprises the steps of:

[0062] (1) DNA template preparation

[0063] Take 5-10 ml of heparin-anticoagulated bone marrow specimens from AML and normal controls, use Ficoll solution to separate mononuclear cells, and use a genomic DNA extraction kit (purchased from Gentra Company) to extract genomic DNA in mononuclear cells. The specific steps are as follows:

[0064] ① Add 1ml TRIZOL (lysate) to the cells and mix well until the water sample;

[0065] ② Add 200 μl of chloroform (chloroform), close the lid tightly, shake and mix well on...

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Abstract

The invention relates to application of a detection substance of an SLC22A3 gene in preparation of a reagent or a medicine for auxiliary diagnosis, treatment effect or prognosis of leukemia. The SLC22A3 gene is shown as SEQ ID NO.1, and the detection substance comprises a forward primer shown as SEQ ID NO.2 and a reverse primer shown as SEQ ID NO.3. The method for detecting leukemia is based on the expression and methylation level of the gene marker SLC22A3 and is combined with other clinical indexes, and more accurate judgment is provided for diagnosis, treatment and prognosis of leukemia.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a detection method and application of molecular markers. Background technique [0002] Acute myeloid leukemia (AML) is a kind of hematopoietic malignant disease originating from myeloid progenitor cells. Its pathogenesis involves abnormal changes in cell differentiation, proliferation, apoptosis, etc. Oncogene mutations play an important role in the occurrence and development of AML. Due to the great heterogeneity of AML, an individualized precise diagnosis and treatment plan based on the patient's biological characteristics on the basis of standardized treatment is urgently needed clinically. In recent years, studies have found that epigenetics changes involving methylation regulation, histone modification, RNA splicing, etc. are another pathological mechanism that plays an important role in the process of leukemia, and demethylation drugs are effective in AML. The curative ef...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154C12Q2600/158
Inventor 谷雨钱军林江马吉春闻向梅徐子浚
Owner ZHENJIANG NO 1 PEOPLES HOSPITAL
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