Osteosarcoma miRNA detection reference gene identification method

A technique for osteosarcoma and internal reference, which is applied in the field of identification of internal reference miRNA genes to achieve the effect of improving sensitivity, specificity and easy detection

Active Publication Date: 2018-05-29
NANJING NOVOACINE BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No studies have systematically identified and evaluated the best reference genes for miRNA normalization in osteosarcoma research. Therefore, there is an urgent need in the field to develop reference genes that can be used as microRNA normalization in osteosarcoma research, and to establish the detection of miRNAs, especially An efficient normalization protocol for circulating miRNAs

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  • Osteosarcoma miRNA detection reference gene identification method
  • Osteosarcoma miRNA detection reference gene identification method
  • Osteosarcoma miRNA detection reference gene identification method

Examples

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Effect test

example 1

[0024] Example 1 Analysis of miRNA expression in peripheral blood of patients with osteosarcoma

[0025] 1. Sample collection

[0026] Peripheral blood of osteosarcoma patients came from hospitalized patients with osteosarcoma, 30 patients with primary osteosarcoma and 20 healthy controls. The patients with primary osteosarcoma and the healthy control group were required to fast for at least 12 hours. At 7:00-8:00 the next morning at room temperature, 10ml of venous blood was drawn into ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes, and peripheral blood was extracted. For mononuclear cell PBMCs, add 1ml Trizol reagent (Invitrogen), mix well, and store the specimen at -80°C for RNA extraction. All blood samples and pathological results should be authentic and reliable, the study was approved by the ethics committee, and the patients gave informed consent.

[0027] 2. Method

[0028] 2.1. Extraction of total RNA from peripheral blood

[0029] According to the in...

Embodiment 2

[0040] The qRT-PCR verification of miRNA in embodiment 2 serum / plasma

[0041] According to the results of chip hybridization, select miRNA molecules that meet the following criteria for further screening by qRT-PCR technology: a) the expression level in osteosarcoma and the control group is in the top 80; b) it is stably expressed in both groups, and the two groups There was no significant difference between them (p≥0.05). According to the above criteria, 6 miRNA molecules (including hsa-miR-128a-5p, let-7d, has-miR-100, has-miR-210-5p, has-miR-222, has- miR-425), the sequences of the six miRNA molecules are shown in Table 1. In addition, since U6 is often used as an internal reference molecule for tissue miRNA detection, U6 was also included in the screening as a candidate molecule in order to verify whether it can be used as an internal reference in serum. By using qRT-PCR technology to verify the above 6 miRNAs and U6 in another group of subjects (including 10 cases of o...

Embodiment 3

[0058] Example 3 Further verification of expression stability

[0059] According to the results of qRT-PCR screening, the combination of has-miR-128a-5p or has-miR-128a-5p / let-7d is a candidate internal reference gene with stable expression. The qRT-PCR method was used to verify its stability in a group of new test samples (including 10 osteosarcoma and 10 control samples), and the results showed that it was stably expressed in both osteosarcoma and normal samples, with relatively good stability.

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Abstract

The invention discloses a reference miRNA gene stably expressed in an osteosarcoma patient. The reference miRNA gene comprises an hsa-miR-128a-5p or combination of hsa-miR-128a-5p and let-7d. The reference gene can be used for standardizing a target gene in osteosarcoma patient miRNA detection, research results of different laboratories can be compared, and normalization processing of experimentalresults is promoted.

Description

technical field [0001] The patent of the present invention relates to the identification of an internal reference miRNA gene for the detection of osteosarcoma miRNA. Background technique [0002] Osteosarcoma (OSA) is a common malignant bone tumor, in which malignantly proliferating sarcoma cells directly produce neoplastic osteoid tissue or immature bone, and its histological feature is that proliferating spindle-shaped tumor cells directly produce osteoid matrix or not. In mature bone, almost all osteosarcoma metastasizes to the lungs via the blood, and a few metastasizes to internal organs such as the brain, kidney, and lymph nodes. The invasion and metastasis of osteosarcoma seriously affect the quality of life and prognosis of patients. Although clinical orthopedic workers have been committed to the research on its prevention and treatment, the overall 5-year survival rate of osteosarcoma has remained stagnant in recent years. Therefore, it is a long way to go to find ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/158C12Q2600/166C12Q2600/178
Inventor 朱伟
Owner NANJING NOVOACINE BIO-TECH CO LTD
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