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Long-chain non-coding RNA MYC-AS1 expression Northern blot detection primer and kit

A RNAMYC-AS1, long-chain non-coding technology, applied in the field of molecular biology

Pending Publication Date: 2020-08-18
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of DNA methylation inhibitors has also caused some confusion

Method used

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  • Long-chain non-coding RNA MYC-AS1 expression Northern blot detection primer and kit
  • Long-chain non-coding RNA MYC-AS1 expression Northern blot detection primer and kit
  • Long-chain non-coding RNA MYC-AS1 expression Northern blot detection primer and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Determination of the full-length sequence of MYC-AS1.

[0026] (1) Identify the 5' and 3' end sequences of MYC-AS1;

[0027] Mainly provided by Takara Corporation RACE 5' / 3'Kit kit to complete.

[0028] First, use Reagent was used to extract total RNA from the human colon cancer cell line HCT116, and then the genome was depleted with RNase-freeDNase I. Under the action of SMARTScribeReverse Transcriptase (provided by the kit), 1 μg of the genome-removed RNA was reverse-transcribed to synthesize 5′- or 3′-RACE products.

[0029] Then, follow Instructions for the operation of the RACE 5' / 3'Kit kit, using the universal primer UPM and 5'-end or 3'-end gene-specific primers (gene-specific primers, GSP) for PCR amplification, cloning and sequencing to obtain MYC- 5' end and 3' end sequences of AS1. Wherein, the nucleotide sequence of the 5'-end or 3'-end gene-specific primer used is as follows:

[0030] MYC-AS1-5-race:

[0031] 5'-CCACAGCAAAACCTCCTCACAGC...

Embodiment 2

[0040] Example 2, MYC-AS1 overexpression vector construction.

[0041] In Example 2, the full-length MYC-AS1 sequence obtained in Example 1 was used to construct a MYC-AS1 overexpression plasmid.

[0042] Specifically include the following steps:

[0043](1) From human colon cancer cell line HCT116, use Reagent was used to extract total RNA, and RNase-free DNase I was used to deplete the genome. It was reverse transcribed into a cDNA product using the PrimeScript RT reagent Kit.

[0044] (2) Using the cDNA product obtained in step (1) of Example 2 as a template, use a high-fidelity enzyme to amplify the full-length sequence of MYC-AS1, wherein the primers MYC-AS1-HindIII-F and MYC-AS1-BamHI- The nucleotide sequence of R is as follows:

[0045] MYC-AS1-HindIII-F:

[0046] 5'-cccaagcttGCCTTTTCATTGTTTTCCA-3' (SEQ ID NO.4)

[0047] MYC-AS1-BamHI-R:

[0048] 5'-cgcggatccCCTTTTTTTAAGACGGAGTC-3' (SEQ ID NO.5)

[0049] The reaction system includes: 100ng of the cDNA product o...

Embodiment 3

[0052] Example 3. Analysis of the ability of MYC-AS1 to inhibit the expression of the proto-oncogene c-myc gene.

[0053] In this Example 3, using the pcDNA3.1-MYC-AS1 overexpression plasmid obtained from Example 2, the ability of MYC-AS1 to inhibit the expression of the proto-oncogene c-myc gene was evaluated by fluorescent quantitative PCR and Western-blot methods .

[0054] Specifically include the following steps:

[0055] (1) Human colon cancer cell line HCT116 cells were inoculated into 6-well cell culture plates, and then transfected with pcDNA3.1-MYC-AS1 overexpression plasmid. Simultaneous transfection of pcDNA3.1-EGFP overexpression plasmid was used as vector control.

[0056] (2) 48 hours after transfection, the cells were collected;

[0057] (3) The total RNA of the cells was extracted, and the expression level of the proto-oncogene c-myc in the cells was detected by a fluorescent quantitative PCR method.

[0058] (4) Extract the total protein of the cells, and...

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to a long-chain non-coding RNA MYC-AS1 expression Northern blot detection primer and a kit. The sequences of the primers are shown as SEQ ID NO. 8 and SEQ ID NO. 9. The invention further provides an MYC-AS1 expression Northernblot detection kit, a method and a basis are provided for analysis of an MYC-AS1 expressionlaw, and the MYC-AS1 expression Northernblot detection kit can be used for detecting the expression situation of oncogenes in a tumor sample in clinical medicine.

Description

technical field [0001] The invention relates to a long-chain non-coding RNA MYC-AS1 expression Northern blot detection primer and a kit, belonging to the field of molecular biology. Background technique [0002] Tumor occurrence is attributed to multiple intrinsic factors, such as genetic mutations, epigenetic variations, and metabolic abnormalities. It is generally believed that there are mainly two types of genes that are closely related to the occurrence of tumors, namely oncogenes and tumor suppressor genes. In normal cells, the expression of oncogenes and tumor suppressor genes maintains a certain balance ratio. Both genetic and epigenetic mutations can cause abnormal expression of oncogenes and tumor suppressor genes, prompting normal cells to transform into cancer cells. [0003] Although genetic mutations are known to be associated with cancer, more and more evidence shows that epigenetic abnormalities are also closely related to the occurrence, development and met...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/178C12Q2600/158
Inventor 崔恒宓胡序明豆春峰陈绪靖
Owner YANGZHOU UNIV
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