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Liquid phase chip for detecting breast cancer prognosis-related gene mRNA expression level

A liquid phase chip and expression level technology, applied in the field of molecular biology, can solve the problems of long process, cumbersome operation, accuracy easily affected by pathological conditions, etc.

Active Publication Date: 2010-12-22
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 transcription, and the detection results are greatly affected by RNA degradation; second, these two methods only use a pair of primers for PCR amplification, which is prone to non-specific binding, resulting in false positives. The positive rate is high; again, these two methods generally only have one housekeeping gene as a control, and their accuracy is easily affected by pathological conditions; defect

Method used

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  • Liquid phase chip for detecting breast cancer prognosis-related gene mRNA expression level
  • Liquid phase chip for detecting breast cancer prognosis-related gene mRNA expression level
  • Liquid phase chip for detecting breast cancer prognosis-related gene mRNA expression level

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: A liquid-phase chip kit for detection of mRNA expression levels of breast cancer prognosis-related genes, mainly including:

[0086] 1. Microspheres coupled with supporting probes (SP)

[0087] The support probe consists of three parts, the 5' end is the amino terminal bound to the microsphere, the 3' end is the specific sequence P1 bound to the support extension probe, the length is 16nt, and the middle is 8 oligonucleotides T Each target gene uses a microsphere and a support probe, and each microsphere has a different fluorescent code. The supporting probes for each target gene are shown in Table 1:

[0088] Table 1 Different target genes and the number of selected microspheres, supporting probes (SP)

[0089] Gene

Microsphere number

SP sequence (5'→3')NH 2 +poly(dT)+P1

P1: SEQID

ESR1

29

NH 2 -TTTTTTTT-GATTTGTAGTAAGATT

1

PGR

45

NH 2 -TTTTTTTT-TTGAGTAATTGAATTG

2

RPLPO

35

N...

Embodiment 2

[0124] Example 2 Detection of samples by using the liquid chip for detection of mRNA expression levels of breast cancer prognosis-related genes described in Example 1

[0125] The formula of described various solutions is as follows:

[0126]

[0127]

[0128] 1. Lysis samples to release total RNA

[0129] 1. Put the sample to be tested 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.

[0130] 2. After digesting the sample for 2 hours, centrifuge at 13,000rpm for 5 minutes at room temperature, and transfer the clear solution in the middle to a new 1.5ml centrifuge tube for later use. (If the solution is still not clear, repeat this step 1 or 2 times.)

[0131] 2. The target mRNA is immobilized on the microspheres by specific binding with the probe

[0132] 1. Take out the probe working solution and thaw at room temperature, then denature at 95°C for 5 minutes, and immediately put it on i...

Embodiment 3

[0164] Example 3: Detection of mRNA expression levels of genes related to breast cancer prognosis Liquid chip detection of ESR1 and PGR (without internal reference genes)

[0165] 1. Design of liquid phase chip preparation (liquid phase chip for detection of mRNA expression levels of ESR1 and PGR)

[0166] Taking the liquid phase chip for detecting the mRNA expression level of ESR1 and PGR as an example, the samples were detected, and the mRNA expression level of ESR1 and PGR in sample 11 was used as a reference standard, defined as 1, and the mRNA expression of ESR1 and PGR in other samples The levels were divided by the mRNA expression levels of ESR1 and PGR in sample 11, and the resulting relative values ​​were compared. Concrete design is as shown in embodiment 1. The synthesis of support probes, support extension probes, amplification extension probes, anti-tag sequence coated microspheres, amplification primers, detection methods, etc. are as described in Example 1 and ...

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Abstract

The invention discloses a liquid phase chip for detecting breast cancer prognosis-related gene mRNA expression level, which mainly comprises a microsphere which aims at different target genes and is coupled to amido modified supporting probes, supporting extended probes connecting the supporting probes and target gene mRNA and amplification extended probes. Each supporting probe mainly comprises a 5' end spacer arm sequence and a 3' end specific sequence P1 which is in complementary pairing with a supporting extended probe; the supporting probe comprises SEQ. ID NO.1 specific to ESR1gene and SEQ. ID NO.2 specific to PGR gene; each supporting extended probe mainly comprises a 5' end specific sequence P2 which can be correspondingly combined with the target genes, a spacer arm sequence and a 3' end specific sequence P3 which is in complementary pairing with the specific sequence P1 of a corresponding supporting probe; and the supporting extended probes comprise amplification extended probes, wherein each amplification extension probe comprises a 5' end specific sequence P4 which can be combined with the target genes, a spacer arm sequence and a 3' end sequence P5; or the liquid phase chip also comprises a labeling probe in complementary pairing with the sequence P5. The liquid phase chip can perform hybridization reaction under the homogeneous reaction condition, and the designed probes have the advantages of high specificity and high signal to noise ratio during detection.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a liquid phase chip for detecting the mRNA expression level of genes related to breast cancer prognosis. Background technique [0002] At present, the treatment methods for breast cancer mainly include endocrine therapy and chemotherapy. Endocrine therapy refers to adjusting the estrogen level in breast cancer patients through drugs or excision of endocrine glands, inhibiting the division of cancer cells, and slowing down the development of tumors. The speed of action is slower than that of chemotherapy. The use of chemical anticancer drugs to treat cancer, referred to as "chemotherapy", includes chemotherapy before, during and after surgery in the treatment of breast cancer, especially postoperative adjuvant chemotherapy is the most commonly used, and its purpose is to eliminate the residual microcancer after surgery lesion. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 许嘉森吴诗杨杨惠夷何嘉英郭元杰
Owner SUREXAM BIO TECH
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