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Quantification method for expression level of WT1 mRNA

An expression level, RT-PCR technology, applied in recombinant DNA technology, microbial determination/inspection, biochemical equipment and methods, etc., can solve complex problems and achieve the effects of simple operation, simple sensitivity, and less labor.

Active Publication Date: 2015-09-23
OTSUKA PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this method, it is necessary to separately measure the expression level of the housekeeping gene (which is used to correct the expression level of the WT1 gene), so it is complicated.

Method used

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  • Quantification method for expression level of WT1 mRNA
  • Quantification method for expression level of WT1 mRNA
  • Quantification method for expression level of WT1 mRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] One-step multiplex RT-PCR assay

[0094] (1) Design of primers and probes

[0095] Human WT1 mRNA is used as the target gene for measurement, GAPDH mRNA is selected as the endogenous control gene (calibration gene) for correcting the expression level of the measurement target, and primers capable of specific amplification and detection of each gene are designed and synthesized groups and probes.

[0096] In order to simultaneously detect the target gene (human WT1 mRNA) and the calibration gene (GAPDH mRNA), fluorescently labeled probes were prepared as follows. That is, the 5' end of the probe for detecting the target gene is labeled with FAM (6-carboxyfluorescein), and the 5' end of the probe for detecting the correction gene is labeled with HEX (6-hexachlorofluorescein). to mark. In addition, the 3' end of each probe was labeled using ATTO-540Q (ATTO-TEC GmbH) as a quenching dye.

[0097] Table 3 shows the sequences of primers and probes used in this example....

Embodiment 2

[0129] Dilution assay using RNA extracted from K562

[0130] In this example, the one-step multiplex RT-PCR method (which simultaneously amplifies WT1 mRNA and GAPDH mRNA) and the two-step RT-PCR method (where the reverse transcription reaction and PCR , separately amplified WT1 mRNA and GAPDH mRNA) were compared in terms of sensitivity. In addition, the two-step RT-PCR method was implemented using the WT1 mRNA measurement kit "Otsuka" (Otsuka Pharmaceutical Co., Ltd.).

[0131] (1) Sequences of primers and probes

[0132] Table 6 shows the sequences of primers and probes used in the WT1 mRNA measurement and GAPDH mRNA measurement by the one-step RT-PCR method. In the two-step RT-PCR method, since the WT1 mRNA measurement kit "Otsuka" was used, the primers and probes were unknown.

[0133] [Table 6]

[0134] Primer sequences and probe sequences (sequence group A) for amplifying WT1 mRNA and GAPDH mRNA

[0135]

[0136] (2) Preparation of standard products

[013...

Embodiment 3

[0150] cross reactivity test

[0151] In this example, the group shown in Table 8 (sequence group B) and the group shown in Table 9 were used as primers and probes, respectively, and a one-step RT-PCR method for simultaneously amplifying WT1 mRNA and GAPDH mRNA was implemented , to evaluate cross-reactivity.

[0152] [Table 8]

[0153] Primer sequences and probe sequences for amplifying WT1 mRNA and GAPDH mRNA

[0154] (sequence group B)

[0155]

[0156] * : indicates the region of the human WT1 gene (NM_024426.4: SEQ ID NO: 1).

[0157] ** : indicates the region of the human GAPDH gene (NM_002046.3: SEQ ID NO: 2).

[0158] [Table 9]

[0159] Primer sequences and probe sequences for amplifying WT1 mRNA and GAPDH mRNA

[0160] (comparison group)

[0161]

[0162] * : indicates the region of the human WT1 gene (NM_024426.4: SEQ ID NO: 1).

[0163] ** : indicates the region of the human GAPDH gene (NM_002046.3: SEQ ID NO: 2).

[0164] As the test samples, Hu...

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Abstract

Provided is a method for quantifying, with ease, in a short time period, and with high sensitivity, human WT1 mRNA expression level which can be used to diagnose cancers such as leukaemia and solid carcinoma, and which can be used to determine bone-marrow transplantation times. This quantification method for human WT1 mRNA expression level uses one-step RT-PCR to quantify human WT1 mRNA expression level, and is characterized in that a reverse transcription reaction and an elongation reaction of human WT1 mRNA and a housekeeping gene (mRNA) are simultaneously and continuously progressed in the same vessel.

Description

technical field [0001] The invention relates to a new method for quantifying the expression level of human WT1 mRNA, which can be used for the diagnosis of leukemia, solid cancer and other cancers and the determination of bone marrow transplantation period. Background technique [0002] The Wilms tumor gene-1 (Wilms tumor gene-1, hereinafter referred to as "WT1") gene was identified by Call et al. in 1990 as a causative gene for Wilms tumor in children (Non-Patent Document 1). Since then, it has been reported that WT1 mRNA is not only expressed at a high level in children's Wilms tumor, but also in solid cancer cell lines (such as gastric cancer cell lines, colorectal cancer cell lines, lung cancer cell lines, and breast cancer cell lines) and other solid cancer cell lines. High-level expression (Non-Patent Document 2). Currently, the WT1 gene is considered to be a cancer-associated gene not only related to children's nephroblastoma but also to various cancers. [0003] Ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/09
CPCC12Q1/6886C12Q2600/16C12Q1/6851C12Q2600/158
Inventor 西条容子伊藤隆太古贺大辅
Owner OTSUKA PHARM CO LTD
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