Targeting medicament curative effect related gene mRNA expressive level detection Luminex and detection method

A technology of expression level and detection solution, which is applied in the field of molecular biology, can solve the problems that the accuracy is easily affected by pathological conditions, the RNA degradation is greatly affected, and the operation is cumbersome, so as to achieve reliable detection results, good specificity, and improve the detection signal Effect

Inactive Publication Date: 2010-05-12
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, RISH is mainly used to analyze the distribution of RNA in tissues or cells to understand the expression of specific genes, and the process is long and cumbersome, so it is not suitable for clinical applications; while reverse transcription quantitative PCR method and real-time fluorescent quantitative PCR method, First of all, both methods require RNA extraction and reverse transcript

Method used

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  • Targeting medicament curative effect related gene mRNA expressive level detection Luminex and detection method
  • Targeting medicament curative effect related gene mRNA expressive level detection Luminex and detection method
  • Targeting medicament curative effect related gene mRNA expressive level detection Luminex and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 A liquid chip kit for detecting the mRNA expression level of genes related to the therapeutic effect of targeted drugs of receptor tyrosine kinase inhibitors mainly includes:

[0059] 1. Microspheres coupled with support probes (SP)

[0060] The support probe is composed of three parts, the 5'end is the amino end that binds to the microsphere, the 3'end is the specific sequence P1 that binds to the support extension probe, the length is 16 nt, the middle is the spacer, the spacer is It is used to separate the support probe from the surface of the microsphere. There is also a spacer sequence inside the support extension probe and the amplification extension probe. By setting an appropriate length of space between the probe sequence and the amino group and inside the probe Arm sequence can reduce steric hindrance, improve the efficiency of hybridization reaction and the specificity of hybridization reaction. The spacer arm of the support probe of the present inventi...

Embodiment 2

[0088] Example 2 Using receptor tyrosine kinase inhibitor targeted drug-related gene mRNA expression level to detect liquid chip detection of clinical samples

[0089] The formulations of the various solutions are as follows:

[0090]

[0091] 1. Lysis the sample to release total RNA

[0092] 1. Scrape the FFPE tumor tissue section from the glass slide, put it into a 1.5ml centrifuge tube, add 300ul homogenization buffer and 3ul proteinase K (50ug / ul), mix well and digest at 65°C for 2h.

[0093] 2. After digesting the sample for 2 hours, centrifuge at 13,000 rpm for 5 minutes at room temperature, and transfer the clear solution to a new 1.5ml centrifuge tube for later use. (If the solution is still not clear, repeat this step 1 or 2 times.)

[0094] 2. The target mRNA is fixed on the microsphere by specific binding with the probe

[0095] 1. Take out the probe working solution and melt it at room temperature, then denature it at 95°C for 5 minutes, and immediately place it on ice.

[009...

Embodiment 3

[0124] Example 3 Detection of mRNA expression levels of genes related to the efficacy of receptor tyrosine kinase inhibitor targeted drugs by liquid chip with different spacer arms

[0125] 1. Design of liquid phase chip preparation (selection of spacer arms) Take the supporting probes of B2M, EGFR, IGF-1R detection liquid chip as examples, and choose different spacer arms respectively. The specific design is shown in Table 8. The synthesis of probes SP, SE and AE, SP sequence coated microspheres, and detection methods are as described in Example 1 and Example 2.

[0126] Table 8 Spacer arm and its length

[0127] Spacer type

length

test group

poly(dT)

8

1

poly(dA)

8

2

(CH2)n

15

3

poly(TTG)

3

4

[0128] 2. Sample detection

[0129] Using the liquid phase chip prepared by the above design, samples 11-15 were detected according to the detection process and method described in Example 2. The detection results are as follows (the data in the table are the detection ...

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Abstract

The invention discloses a receptor tyrosine kinases inhibiting type targeting medicament curative effect related gene mRNA expressive level detection Luminex. The detection Luminex mainly comprises a micro-ball, supporting extending probes, and amplification extension probes. The micro-ball is coupled with a supporting probe modified by amino; the supporting extending probes are used for connecting the supporting probe and a target gene mRNA, and each supporting extending probe comprises a specific sequence the terminal 5' of which can combine with a corresponding target gene mRNA, a spacer arm sequence, and a sequence the terminal 3' of which can complementarily pair with the corresponding specific sequence of the supporting probe at; and the amplification extension probes are used for connecting the target gene mRNA and information detection components, and each amplification extension probe comprises a specific sequence the terminal 5' of which can combine with the corresponding target gene mRNA, a spacer arm sequence, and a sequence the terminal 3' of which can complementarily pair with a markup probe. The invention also discloses a detection method of the receptor tyrosine kinases inhibiting type targeting medicament curative effect related gene mRNA expressive level by using the liminex. In the invention, the process can be carried out without the steps of RNA extraction, reverse transcription, PCR and the like and the detection result is influenced less by the quality of RNA in the sample, thereby the accuracy of the detection result is guaranteed; on the other hand, because the liminex technology enables a parallel multi-index detection to be realized, multiple inside genes can be set in one detection, thereby the detection result is more reliable.

Description

Technical field [0001] The invention belongs to the field of molecular biology, and relates to medicine and biotechnology, and specifically relates to a liquid chip and a detection method for detecting the mRNA expression level of genes related to the therapeutic effect of receptor tyrosine kinase inhibitor targeted drugs. Background technique [0002] Tyrosine kinase occupies a very important position in the intracellular signal transduction pathway, regulating a series of physiological processes such as cell growth, differentiation, and death, and its abnormal expression will lead to the disorder of cell proliferation regulation, which will lead to tumorigenesis. In addition, the abnormal expression of tyrosine kinase is also closely related to tumor invasion and metastasis, tumor angiogenesis, and tumor resistance to chemotherapy. Drug development with tyrosine kinase as the target has become an international anti-tumor drug research Hot spot. [0003] Tyrosine kinases include ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 许嘉森郭元杰罗彩英吴诗扬杨惠夷
Owner SUREXAM BIO TECH
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