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Kit for detecting IncRNA-NEAT1 in serum and application thereof in liver cancer serological diagnosis

A detection kit and detection reagent technology, applied in the field of medical biological detection, can solve the problems that the value of lncRNA-NEAT1 tumor serological diagnostic markers has not yet been reported in the literature

Active Publication Date: 2015-06-24
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The value of lncRNA-NEAT1 as a tumor serological diagnostic marker has not been reported in the literature so far

Method used

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  • Kit for detecting IncRNA-NEAT1 in serum and application thereof in liver cancer serological diagnosis
  • Kit for detecting IncRNA-NEAT1 in serum and application thereof in liver cancer serological diagnosis
  • Kit for detecting IncRNA-NEAT1 in serum and application thereof in liver cancer serological diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Screening and identification of lncRNA-NEAT1

[0046] In the early stage, we used a high-throughput lncRNA chip to screen lncRNAs related to liver cancer stem cells, and selected 10 lncRNAs with the most significant changes from the screened lncRNAs, and then compared the lncRNAs in liver cancer stem cells enriched from Huh7 liver cancer cells. The expression was verified by PCR, and it was found that lncRNA-NEAT1 expressed the highest in liver cancer stem cells (see figure 1 ).

[0047] It is currently believed that tumors originate from tumor stem cells, so these results suggest that lncRNA-NEAT1 may play a role in the early stages of malignant tumors including liver cancer, and also suggest that it may have a certain application value in tumor diagnostic markers.

Embodiment 2

[0048] Example 2: Collection and processing of serum samples and extraction of small RNAs therein

[0049] (1) Serum sample collection: Collect about 4ml of peripheral blood from patients with hepatocellular carcinoma before operation, put it into an anticoagulant tube containing EDTA, and let it stand for about 1 hour.

[0050] (2) Serum sample processing: the collected peripheral blood samples were centrifuged at 4000 rpm at 4°C for 10 min, the supernatant was taken and packed into EP tubes, and stored at -80°C.

[0051] (3) Extraction of small RNAs in serum: 350 μl of each serum sample was taken, and according to the mirVana purchased from Ambion, TM PARIS TM Kit to extract small RNA.

[0052] (4) Reverse transcription into cDNA: take the same amount of RNA from each sample, and perform reverse transcription with random primer N6. According to 5×M-MLV RT Buffer 5 μl, M-MLV Reverse transcriptase 1 μl, rRNasin 0.5 μl (Promega Company), dNTPs 1 μl, random primer N61 μl, ext...

Embodiment 3

[0053] Example 3: Primer Design and Screening

[0054] (1) Primer design: The full-length sequence of lncRNA-NEAT1 (NR_028272.1) was found by NCBI, and 8 pairs of upstream and downstream primers for Real-time PCR were designed using Primer Premier 5 software for the sequence of lncRNA-NEAT1 (as listed in Table 1. Shown), Invitrogen was responsible for the synthesis of primers.

[0055] Table 1: Eight pairs of primers designed to detect lncRNA-NEAT1 in serum

[0056]

[0057]

[0058] (2) Primer screening: SYBR Premix Ex Taq from TaKaRa Company TM Reagents and the ViiA7Dx real-time quantitative PCR instrument of Applied Biosystems in the United States were used to carry out the PCR reaction according to the following system:

[0059]

[0060] PCR conditions:

[0061]

[0062] According to the above Real-time PCR conditions, 8 pairs of lncRNA-NEAT1 primers were detected by using the reverse transcription products of 3 previously prepared serum samples. Two replic...

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Abstract

The invention relates to the technical field of biomedical detection. By using an IncRNA chip to screen the IncRNA related to a tumor stem cell, the result shows that IncRNA-NEAT1 is highly expressed in the liver cancer stem cell and the liver cancer stem cell function can be adjusted. The invention discloses an application of the IncRNA-NEAT1 in preparation of a liver cancer diagnostic marker and an application of the IncRNA-NEAT1 in preparation of a detection reagent or a kit for diagnosing the liver cell. Moreover, the invention discloses a method for detecting the IncRNA-NEAT1 in a human serum and a kit for detecting the IncRNA-NEAT1 in the serum. Through the detection of the human serum of a liver cancer patient, the IncRNA-NEAT1 can be detected in the serum and the IncRNA-NEAT1 content in the serum of an HCC patient is obviously higher than a normal person. The kit provides a novel clinical solution for the liver cancer serological diagnosis.

Description

technical field [0001] The invention relates to the technical field of medical biological detection, in particular to a kit for detecting lncRNA-NEAT1 in serum and its application in serological diagnosis of liver cancer. Background technique [0002] Long non-coding RNA (long non-coding RNA, lncRNA) is a non-coding RNA with a length greater than 200 nucleotides, its expression has obvious tissue and cell specificity, and its mode of action is diverse. Gene expression is regulated at multiple levels including translation. lncRNA can widely participate in life processes such as cell differentiation and individual development, and its abnormal expression is closely related to many major human diseases including tumors. [0003] lncRNA-NEAT1 (nuclear paraspeckle assembly transcript 1, nuclear paraspeckle assembly transcript 1) includes two transcripts, 3.7kb NEAT1v1 (NR_028272.1) and 23kb NEAT1v2 (HG503867.1), both of which are nuclear paraspeckles important parts of. Nuclea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/158C12Q2600/178
Inventor 王红阳丁劲王林辉陈程曲乐李晓峰孙文王雪刘娜
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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