Quantification method for expression level of WT1 mRNA

Patent metadata

Field	Value
Publication number	US-10280467-B2
Application number	US-201414762454-A
Country	US
Kind code	B2
Filing date	Jan 22, 2014
Priority date	Jan 22, 2013
Publication date	May 7, 2019
Grant date	May 7, 2019

Abstract

A method for quantifying the expression level of human WT1 mRNA conveniently, in a short period of time, and with high sensitivity is provided. The method can be used for diagnosing cancer, such as leukemia and solid cancer, or for determining when to perform bone marrow transplantation. The method is for quantifying the expression level of human WT1 mRNA by one-step RT-PCR and comprises simultaneously subjecting the human WT1 mRNA and a housekeeping gene (mRNA) to reverse transcription and extension reactions carried out sequentially in the same vessel.

First claim

The invention claimed is:

1. A method for quantifying the expression level of human WT1 mRNA in a test sample by one-step reverse transcription PCR, the method comprising simultaneously subjecting the human WT1 mRNA and a housekeeping gene to reverse transcription and extension reactions carried out sequentially in the test sample in the same vessel,
wherein the housekeeping gene is GAPDH mRNA;
wherein the following (a) and (c-1) are used for PCR amplification of the human WT1 mRNA and the housekeeping gene, respectively, or the following (b) and (c-2) are used for PCR amplification of the human WT1 mRNA and the housekeeping gene, respectively:
(a) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 3 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 4, and
(c-1) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 6 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 7, or
(b) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 9 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 10, and
(c-2) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 6 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 12.

2. The method according to claim 1 , wherein the following (a′) and (c′-1) are used for PCR amplification of the human WT1 mRNA and the housekeeping gene, respectively, or the following (b′) and (c′-2) are used for PCR amplification of the human WT1 mRNA and the housekeeping gene, respectively:
(a′) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 3 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 4, and a probe consisting of the base sequence set forth in SEQ ID NO: 5, the probe being labeled, and
(c′-1) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 6 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 7, and a probe consisting of the base sequence set forth in SEQ ID NO: 8, the probe being labeled,
or
(b′) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 9 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 10, and a probe consisting of the base sequence set forth in SEQ ID NO: 11, the probe being labeled, and
(c′-2) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 6 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 12, and a probe consisting of the base sequence set forth in SEQ ID NO: 8, the probe being labeled.

3. A kit for reverse transcription real-time PCR for quantifying the expression level of human WT1 mRNA, the kit comprising the following (a), (c-1) and (d), or (b), (c-2) and (d):
(a) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 3 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 4, and a sequence-specific binding probe used for detecting amplification product amplified by the primer set, the probe being labeled,
(c-1) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 6 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 7, and a sequence-specific binding probe used for detecting amplification product amplified by the primer set, the probe being labeled, and
(d) at least one component selected from the group consisting of dNTPs, Mg salts, buffering components for pH adjustment, enzymes having reverse transcription activity, components for stabilizing an enzyme, ribonuclease inhibitors, and a reaction vessel, or
(b) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 9 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 10, and a sequence-specific binding probe used for detecting amplification product amplified by the primer set, the probe being labeled, and
(c-2) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 6 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 12, and a sequence-specific binding probe used for detecting amplification product amplified by the primer set, the probe being labeled, and
and the above-mentioned (d).

4. A kit for reverse transcription real-time PCR for quantifying the expression level of human WT1 mRNA, the kit comprising the following (a′), (c′-1) and (d), or (b′), (c′-2) and (d):
(a′) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 3 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 4, and a probe consisting of the base sequence set forth in SEQ ID NO: 5, the probe being labeled,
(c′-1) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 6 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 7, and a probe consisting of the base sequence set forth in SEQ ID NO: 8, the probe being labeled, and
(d) at least one component selected from the group consisting of dNTPs, Mg salts, buffering components for pH adjustment, enzymes having reverse transcription activity, components for stabilizing an enzyme, ribonuclease inhibitors, and a reaction vessel, or
(b′) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 9 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 10, and a probe consisting of the base sequence set forth in SEQ ID NO: 11, the probe being labeled,
(c′-2) a primer set comprising a forward PCR primer consisting of the base sequence set forth in SEQ ID NO: 6 and a reverse PCR primer consisting of the base sequence set forth in SEQ ID NO: 12, and a probe consisting of the base sequence set forth in SEQ ID NO: 8, the probe being labeled, and
the above-mentioned (d).

Assignees

• Otsuka Pharma Co Ltd

Inventors

• Saijo Yoko
• Ito Ryuta
• Koga Daisuke

Classifications

• C12Q1/6851

  Quantitative amplification · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/16

  Primer sets for multiplex assays · CPC title