mRNA in-situ hybridization kit for detecting overexpression of her2

A detection kit and in-situ hybridization technology, which is applied in the field of RNA in-situ hybridization detection kits to achieve the effects of overcoming tissue endogenous biotin interference, enhancing specificity and preventing competitive hybridization

Inactive Publication Date: 2011-06-15
上海裕隆医学检验所股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] At present, there is no product on the market that use...

Method used

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  • mRNA in-situ hybridization kit for detecting overexpression of her2
  • mRNA in-situ hybridization kit for detecting overexpression of her2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Primer design and probe synthesis

[0042] Design specific primers for RT-PCR according to the nucleotide sequence of HER2 gene (NO.M11730), the sequence is as follows:

[0043] The HER2 forward primer sequence is: GATGCCCAACCAGGCGCAGAT 21bp;

[0044] The sequence of the HER2 reverse primer is: ACCAGCTGCACCGTGGATGTCAG 23bp.

[0045] Using the human blood RNA extracted by the Trizol method as a template for RT-PCR amplification, the probe-specific fragments are obtained, as shown in the figure: the electrophoresis diagram of the HER2 probe fragment amplified by RT-PCR; the specificity obtained by RT-PCR amplification The sequence is as follows:

[0046] GATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTTTGGCACAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGTCGTGCGTGCGTGAAGGTGCTTGGATCTGGCGGTGCGTGCAGTCTACAAGGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCTGGCGCTGG...

Embodiment 2

[0056] Example 2: Reagent preparation

[0057] 1. The fixative solution is 4% paraformaldehyde (pH 7.4) prepared in RNase-free PBS.

[0058] 2. PBS-Buffer I is made of 140mmol / LNaCl, 2.7mmolKCl, 10mmol / LNa treated with DEPC 2 HPO 4 And 1.8mmol / L KH 2 PO 4 Composition of pH7.4 buffer.

[0059] 3. PBS-Buffer II PBS-Buffer I containing 100mmol / L glycine.

[0060] 4. PBS-Buffer III PBS-Buffer I containing 0.3% TritonX-100.

[0061] 5. Digestion solution A 20μg / ml proteinase K solution without RNase prepared by TE buffer (100mmol / L Tris-HCl, 50mmol / LEDTA, pH8.0).

[0062] 6. The pre-hybridization solution is a solution composed of 50% deionized formamide, 20×SSC, 50×Denhardt, and 0.1 mg / ml tRNA.

[0063] 7. Washing Buffer I is a solution containing 300mol / L NaCl and 30mol / L sodium citrate (pH7.0).

[0064] 8. Washing Buffer II is a solution containing 150mmol / L NaCl and 15mmol / L sodium citrate (pH7.0).

[0065] 9. Buffer I is a solution containing 100 mmol / L Tris-HCl and 150 mmol / L NaCl (pH 7.5)...

Embodiment 3

[0071] Example 3: Use of the kit

[0072] 1. Tissue section processing

[0073] 1) Dewaxing xylene at 37℃ twice, 15 minutes each time;

[0074] 2) Soak in absolute ethanol twice, 3 minutes each time;

[0075] 3) Soak in 95% ethanol twice, 3 minutes each time;

[0076] 4) Wash with PBS-Buffer I twice, 3 minutes each time;

[0077] 5) Wash twice with PBS-Buffer II, 3 minutes each time;

[0078] 6) Add digestion solution and incubate at 37°C for 20 minutes;

[0079] 7) Wash twice with PBS-BufferIII, 5 minutes each time;

[0080] 8) Fixing solution treatment for 10 minutes;

[0081] 9) Wash twice with PBS-Buffer I, 5 minutes each time;

[0082] 2. Pre-hybrid

[0083] Add pre-hybridization solution and incubate at 50°C for 1 hour;

[0084] 3. Hybrid

[0085] Add hybridization solution and incubate overnight at 50°C.

[0086] 4. Elution

[0087] 1) Washing Buffer I wash 2 times, 15 minutes each time;

[0088] 2) Washing Buffer II twice, 15 minutes each time;

[0089] 3) Buffer I treatment 2 times, 5 minut...

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Abstract

The invention discloses an RNA in-situ hybridization (ISH) kit for detecting overexpression of human epidermal growth factor receptor (EGFR) 2 (HER2) genes of breast cancer patients and belongs to the field of molecular pathological diagnosis. The kit comprises stationary solution, PBS-BufferI, PBS-BufferII, PBS-BufferIII, digestive juice, prehybridization solution, hybridization solution, Washing BufferI, Washing BufferII, buffer solution I, buffer solution II, blocking liquid, development solution I, development solution II, and a positive control tissue glass plate. Reference basis is provided for diagnosing positive breast cancer patients and important clinical value for early diagnosis of breast cancer and formulation of personalized medicines is provided by detecting the expression level of HER2.

Description

Technical field [0001] The invention belongs to the field of molecular pathological diagnosis, and particularly relates to an RNA in situ hybridization (ISH) detection kit for detecting the overexpression of human epidermal growth factor receptor 2 (HER2) gene in breast cancer patients in clinical samples. Background technique [0002] Breast cancer is a serious threat to people’s health. The incidence of breast cancer in almost all populations is increasing, with an average annual increase of about 1%. It is estimated that more than 1 million patients are affected worldwide each year. The Ministry of Health issued the “Third National "Main Causes of Death Investigation" shows that the incidence and mortality of breast cancer are on the rise all over the world, and the population is getting younger and younger. According to the data of the World Health Organization: About 1.4 million women worldwide suffer from breast cancer each year, and 400,000 women die from breast cancer. I...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 穆海东汪宁梅穆宇豪黎飒
Owner 上海裕隆医学检验所股份有限公司
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