Twist gene expression detection reagent kit

A detection kit and gene expression technology, applied in the field of molecular biology, can solve the problems of high experimental conditions, limited sensitivity, difficulty in ensuring accurate and reliable detection of gene expression levels, etc., to improve spatial distribution density, signal-to-noise The effect of high ratio and reduced probability of non-specific binding

Pending Publication Date: 2020-03-27
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method has the advantages of high sensitivity and strong sequence specificity; however, this method has high requirements for experimental conditions. From the beginning of sampling to the analysis of final results, every step must be strictly operated, otherwise it is difficult to ensure the detection of gene expression level results Accurate and reliable (Bustin S&Nolan T, European journal of clinical investigation, 2017, 47, 756-774)
Patent CN102925543A discloses an in situ hybridization detection kit, detection method and application of pre-leukemia lesion mRNA level. Hybridization technology can detect the expression of Twist gene at the mRNA level, which is convenient and simple to operate, but its sensitivity is limited to a certain extent due to the inability to amplify the signal

Method used

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  • Twist gene expression detection reagent kit
  • Twist gene expression detection reagent kit
  • Twist gene expression detection reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Twist gene expression detection kit

[0043] The Twist gene expression detection kit described in this embodiment mainly includes:

[0044] 1. False Capture Probes

[0045] False capture probes are combinations of local sequences containing different repeats of human transposons that neither hybridize to target mRNA nor to capture probes, amplification probes, and labeled probes with terminally modified fluorescent groups. The needles hybridize. The false capture probe is used for the pre-hybridization treatment of the biological sample to be detected, which can effectively prevent the non-specific binding of the capture probe to the non-target mRNA in the biological sample to be detected, and improve the specificity of detection (for specific use, select at least 2 The purpose of improving the specificity of detection can be achieved by using the false capture probe, for which reference may be made to Example 6 and Example 7). In this embodiment, three fal...

Embodiment 2

[0063] Example 2 Using the kit in Example 1 to detect the sample

[0064] The formula of described various solutions is as table 6:

[0065]

[0066] In this embodiment, the blood samples of tumor patients are preferred, and the expression level of Twist gene of circulating tumor cells (CTCs) in the samples is detected, wherein the pre-capture mixed solution, capture mixed solution, amplification mixed solution, and chromogenic mixed solution are all used in Example 1. All probes in the corresponding list of the Twist Gene Expression Assay Kit.

[0067] 1. Sample pretreatment, the cells to be tested are filtered onto the filter membrane

[0068] (1) Collect the cell suspension to be tested, centrifuge horizontally at 600×g for 5 minutes, and discard the supernatant. (2) Add 4ml of PBS and 1ml of fixative, vortex and mix well, and let stand at room temperature for 8min. (3) Sample filtration: transfer the liquid to the filter, turn on the vacuum pump to pump out the liqui...

Embodiment 3

[0098] Example 3 The impact of capture probe structure on the detection effect of the kit

[0099] 1. Design of kit preparation (capture probe structure design)

[0100] In order to evaluate the detection effect of kits composed of capture probes with different structures, experimental groups 1-3 were designed, and the three groups were all the same except for different capture probes. Among them, the experimental group 1 selected the self-assembled polymer branched nucleic acid probe in the kit of the present invention; the experimental group 2 selected the linear capture probe, and its composition structure from the 5' end to the 3' end is as follows: α1 or α2 region sequence, β1 or β2 region sequence and the γ region sequence composed of the specific sequence P1, spacer sequence and P2 sequence that can bind to the target mRNA to be detected, the base of the α1 or α2 region sequence, β1 or β2 region sequence and the γ region sequence The base composition is exactly the sam...

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Abstract

The invention provides a Twist gene expression detection reagent kit. The reagent kit comprises a false catching probe for detection of Twist gene mRNA, a catching probe and a signal amplification system, wherein the signal amplification system comprises an amplification probe and a labelling probe; the false catching probe is used for prehybridization of a biological sample, and the catching probe is a self-assembled polymerized and branched nucleic acid probe and is connected with target mRNA and the amplification probe, the amplification probe is used for being connected with the catching probe and the labelling probe, and the labelling probe is connected with the amplification probe and fluorophore. The reagent kit has the advantages of being high in sensitivity, high in specificity, high in accuracy rate and the like, and can be used for detecting the expression level of the Twist gene in the biological samples.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a Twist gene expression detection kit. Background technique [0002] Twist gene, one of the members of the basic helix-loop-helix (bHLH) transcription factor family, was first discovered in Drosophila in 1987, and it plays a key regulatory role in embryonic development (Pei H, et al., MedChemComm, 2017, 8, 268-275). So far, two Twist genes have been identified in vertebrates, namely Twist 1 and Twist 2 (also known as Dermo-1), and their bHLH and carboxy-terminal functional domains have more than 90% similarity; however, the Twist 1 The amino terminus is rich in glycine, whereas the amino terminus of Twist 2 lacks glycine. Wherein, Twist 1 is the so-called Twist gene. The human Twist gene is located on chromosome 7p21.1 and contains 4 exons. Twist is rarely detected in healthy adult tissues, but is often highly expressed in various cancer tissues such as breast cancer, prostat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/682C12Q1/6841
CPCC12Q1/6886C12Q1/682C12Q1/6841C12Q2600/158C12Q2600/118C12Q2600/166C12Q2563/107C12Q2537/162
Inventor 许嘉森吴诗扬彭璨璨刘志明刘芳
Owner SUREXAM BIO TECH
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