Kit for detecting gene expression of cytokeratin 18

A detection kit and gene expression technology, applied in the field of molecular biology, can solve problems such as high requirements for experimental conditions, difficulty in ensuring the accuracy and reliability of detection results, and limited tissue samples required for detection, so as to improve detection Efficiency, improvement of detection effect, shortening the effect of amplification and hybridization time

Pending Publication Date: 2021-08-10
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Immunohistochemistry detects the expression of CK18 at the protein level; however, the limitations of this method are: the tissue samples required for the detection are limited, and the accuracy of the detection results is limited by the type of antibody, antigen retrieval method, staining method and conditions, etc. Influenced by factors, result interpretation is highly subjective
Real-time quantitative PCR is mainly used to detect the expression level of CK18 gene in tissue / cell samples, which has the advantages of high sensitivity and strong sequence specificity; however, this method has high requirements on experimental conditions, and contamination must be strictly avoided, otherwise it is difficult to guarantee the detection results accuracy and reliability
In view of the above problems, Chinese patent CN201410228511.9 provides an RNA in situ hybridization method for detecting gene expression. The detection probe of the method can realize the fluorescence signal amplification of RNA in situ detection and improve the sensitivity of detection, but further research It was found that the hybridization selectivity of the capture probe and the signal amplification system in the above RNA in situ hybridization detection method need to be further optimized to achieve better detection results

Method used

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  • Kit for detecting gene expression of cytokeratin 18
  • Kit for detecting gene expression of cytokeratin 18
  • Kit for detecting gene expression of cytokeratin 18

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 A kind of cytokeratin 18 gene expression detection kit

[0051] A cytokeratin 18 gene expression detection kit described in this embodiment mainly includes:

[0052] 1. Capture Probes

[0053] From the 5' end to the 3' end of the capture probe, there are a stem structure sequence, a specific P1 sequence capable of binding to the target mRNA to be detected, a linker (Linker), and a P2 sequence. The P2 sequences in the capture probes of the same target mRNA are the same. The stem sequence is a γ-modified peptide nucleic acid (γPNA) sequence with a base length of 4 to 8 bp, which can be complementary to the base at the 3' end of the P2 sequence to form a stem-loop structure; the specific P1 sequence is a base length γPNA sequence of 10-22bp; the Linker is EG 8 Linker; the P2 sequence has a base length of 8-20 bp, no hairpin structure, no dimer formation or mismatch within the probe and between probes, and no specificity between P1 and target mRNA sex-bindi...

Embodiment 2

[0081] Example 2 Using the kit described in Example 1 to detect the sample

[0082] The formula of described various solutions is as table 7:

[0083] Formulation of various solutions of table 7

[0084]

[0085]

[0086] In this embodiment, the blood samples of tumor patients are preferred, and the expression level of CK18 gene of circulating tumor cells in the samples is detected, wherein the capture mixed solution, the chromogenic mixed solution SP1, and the chromogenic mixed solution SP2 all use all of the corresponding lists described in Example 1. probe.

[0087] 1. Take 5ml of blood from the patient's vein into a vacuum blood collection tube to obtain a blood sample.

[0088] 2. Sample pretreatment, the cells to be tested are filtered onto the filter membrane.

[0089] (1) Collect the cell suspension to be tested, centrifuge at 600×g for 5 minutes, and discard the supernatant; (2) Add 4ml PBS and 1ml fixative, vortex and mix, and let stand at room temperature f...

Embodiment 3

[0111] Embodiment 3 The impact of different types of capture probes on the detection effect of the kit

[0112] 1. Design of kit preparation (capture probe design)

[0113] In order to evaluate the detection effect of kits composed of different types of capture probes, experimental groups 1-3 were designed, and each group had the same components except for the types of capture probes. The specific design is shown in Table 10.

[0114] Table 10 Selection of kit capture probes

[0115]

[0116]

[0117] 2. Sample detection

[0118] In this example, commercially available cell lines NCl-H1975 and CCRF-HSB-2 were selected for experiments. Take 3000 NCl-H1975 and CCRF-HSB-2 cells respectively (determined by a cell counter), and after mixing, divide each sample into 15 parts, numbered 1-15 and 16-30 in sequence. Using the kit prepared by the above design, samples 1 to 30 were detected according to the detection process and method described in Example 2, and 5 samples of ea...

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Abstract

The invention relates to a cytokeratin 18 gene expression detection kit and a detection method thereof. The cytokeratin 18 gene expression detection kit comprises a capture probe aiming at CK18 gene mRNA and a signal amplification system, the capture probe is a stem-ring-shaped gamma PNA-PNA probe, which sequentially comprises a stem structure sequence, a P1 sequence, a Linker sequence and a P2 sequence from the 5' end to the 3' end; and the signal amplification system comprises a marking probe SP1 and a marking probe SP2, wherein each labeled probe SP1 sequentially comprises a P3 sequence, a spacer sequence and a P4 sequence from the 5' end to the 3' end; the 5' end of the P3 sequence is modified with a fluorophore, each labeled probe SP2 sequentially comprises a P5 sequence, a spacer sequence and a P6 sequence from the 5' end to the 3' end, and the 3' end of the P6 sequence is modified with a fluorophore. The kit has the advantages of high sensitivity, strong specificity, high accuracy, short detection time and the like.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a cytokeratin 18 gene expression detection kit. Background technique [0002] Cytokeratin 18 (CK18) is a type I acidic intermediate filament protein belonging to the cytoskeleton protein family. Its gene is located on human chromosome 12q13.13 and contains 8 exons. CK18 is mainly expressed in monolayer or "simple" epithelial tissues, such as liver, lung, kidney, pancreas, gastrointestinal tract, breast, etc., and is also expressed in cancers arising from these tissues (WengYR et al., MolCancer Res, 2012, 10:485–493). The known biological function of CK18 is to provide a flexible intracellular scaffold to build the cytoplasm, resist external pressure on the cell, and maintain normal mitochondrial structure (WengYR et al., Mol Cancer Res, 10:485–493) . CK18 also plays an important role in processes such as apoptosis, mitosis, cell cycle progression and cell signal transduction. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/682C12Q1/6886C12N15/11
CPCC12Q1/682C12Q1/6886C12Q2600/158C12Q2525/107C12Q2563/107
Inventor 许嘉森吴诗扬黄洁芬
Owner SUREXAM BIO TECH
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