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Method and kit for testing rabbit bone morphogenic protein-2 genes

A kit and morphological technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of experimental repeatability and poor reliability, and achieve short experimental cycle, high sensitivity and step-by-step simple effect

Active Publication Date: 2010-07-14
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004]At present, the detection of gene expression level is mainly done by polymerase chain reaction (Polymerase Chain Reaction, PCR), which is an experiment widely used in molecular biology Technology, through the thermostable Taq DNA polymerase, the single or several copies of the gene fragments in the template are exponentially amplified after dozens of thermal cycles, reaching millions of copies, which is currently commonly used in gene expression Compared with the quantitative detection method, the expression of the target gene is detected by electrophoresis of the PCR product. Since the progress of the reaction cannot be monitored in real time, the repeatability and reliability of the experiment are relatively poor.

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  • Method and kit for testing rabbit bone morphogenic protein-2 genes
  • Method and kit for testing rabbit bone morphogenic protein-2 genes
  • Method and kit for testing rabbit bone morphogenic protein-2 genes

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Embodiment 1

[0045] The GENEBANK accession number of the rabbit bone morphoprotein 2 gene is gi-2773387, and the real-time quantitative PCR primers and probes of the rabbit bone morphoprotein 2 gene and the rabbit GAPDH gene were designed by using the primer design software Beacon designer 7, and were analyzed and excluded by the software Beacon designer 7. The inter-primer / internal dimer and hairpin structures were determined, and the specificity of the primers and the homology with similar genes were verified by BLAST, and the following primers and probes were designed.

[0046] Rabbit bone morphoprotein 2-specific primers are divided into upstream primers and downstream primers:

[0047] Upstream primer sequence: 5'-GAAATTCCCCGCCACCAGAC-3' (its nucleotide sequence is shown in SEQ ID NO.1)

[0048] Downstream primer sequence: 5'-TCACTTCCACCACAAACCCATG-3' (its nucleotide sequence is shown in SEQ ID NO.2)

[0049] The specific Taqman probe sequence of rabbit bone morphoprotein 2 is 5'6-FA...

Embodiment 2

[0058] The kit of the present invention consists of: 1.25ml 10×PCR Buffer, 200μl of 2μM ROX, 1ml of 10mM dNTPs, 250μl of 5μM upstream primer, 250μl of 5μM downstream primer, 250μl of 5μM fluorescently labeled Taqman probe, Taq Enzyme 500U, nuclease-free water 2ml, standard (1010 copies) 20μl.

[0059] The primers include the upstream primer and the downstream primer of the rabbit bone morphoprotein 2 prepared in Example 1, the upstream primer and the downstream primer of the rabbit GAPDH gene; the probes include the probe of the rabbit bone morphoprotein 2 and the probe of the rabbit GAPDH gene. Needle.

[0060] The reference substance is divided into negative control and positive control, the negative control is nuclease-free water, and the positive control is the cDNA sample of rabbit muscle tissue.

[0061] It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications...

Embodiment 3

[0063] Surgical amputation of the radius of the rabbit's foreleg resulted in a 2cm bone defect. The bone defect was implanted with pure PLGA and HA and PLGA blends with different grafting ratios. The mass percentages of HA and PLGA in the blend were 80% and 20%, respectively. The grafting ratios of grafted HA were 1%, 3%, 5%, 7%, 10%, respectively. The bone repair materials used in animal experiments were PLGA, PLGA+HA, PLGA+HA (1%), PLGA+HA (3%), PLGA+HA (5%), PLGA+HA (7%), PLGA+HA ( 10%). Three months after the operation, take out the material from the bone defect site, weigh 100 mg, add liquid nitrogen to the mortar and mash, use Trizol to extract the total RNA in the material, take 1 μg of total RNA for reverse transcription into cDNA, and use these cDNA to separate Real-time PCR reaction was performed with BMP-2 primers and GAPDH primers, and 3 parallel samples were set up for each sample. In the experiment, GAPDH was set as the internal reference group to correct the e...

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Abstract

The invention relates to the technical field of biology, in particular to a method for testing rabbit bone morphogenic protein-2 genes. The method includes the following steps that: an upstream primer with a nucleotide sequence shown by SEQ ID NO.1 and a downstream primer with a nucleotide sequence shown by SEQ ID NO.2 are used to amplify biological sample, and a probe with a sequence shown by SEQ ID NO.3 is used to carry out testing. The invention also provides a kit for testing rabbit bone morphogenic protein-2 genes. The method has high sensitivity in testing rabbit bone morphogenic protein-2 genes, the specificity is strong, the operation is simple, the sample range is wide, and thereby, the method has a broad application prospect.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method and a kit for detecting rabbit bone morphoprotein 2 gene. Background technique [0002] Bone morphogenetic protein is a group of cytokines that can induce bone and cartilage formation. BMP binds to BMP receptors on the cell surface, causing cell signal transduction and activating SMAD family proteins. Seven bone morphoproteins were initially discovered, among which bone morphoproteins 2-7 belong to TGF-β family proteins, and bone morphoprotein 1 belongs to metallothionein. At present, 20 bone morphoproteins have been discovered, and two types of bone morphoproteins, bone morphoprotein 2 and bone morphoprotein 7, have been certified by the US FDA. [0003] BMP2, like other BMPs, plays an important role in the development of bone and cartilage. BMP2 has osteogenic ability and can induce the differentiation of osteoblasts into other types of cells. Adding...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64C12N15/11
Inventor 章培标王宇陈学思王宗良崔立国
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI