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Long noncoding RNA, primer pair for detecting expression level of long noncoding RNA in cell line and tissue as well as kit

A long-chain non-coding, primer pair technology, used in DNA/RNA fragmentation, recombinant DNA technology, microbial determination/inspection, etc., can solve problems such as unclear functions, and achieve simple operation, high sensitivity, and good stability. Effect

Active Publication Date: 2015-09-02
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More and more long non-coding RNAs have been discovered, but their functions are still unclear

Method used

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  • Long noncoding RNA, primer pair for detecting expression level of long noncoding RNA in cell line and tissue as well as kit
  • Long noncoding RNA, primer pair for detecting expression level of long noncoding RNA in cell line and tissue as well as kit
  • Long noncoding RNA, primer pair for detecting expression level of long noncoding RNA in cell line and tissue as well as kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Template preparation (total RNA extraction and reverse transcription process)

[0031] Preparation of RNA: Colon cancer tissue and paired paracancerous tissue samples, gastric cancer tissue and paired paracancerous tissue samples were obtained. In this embodiment, 8 pairs of colon cancer tissues and paired paracancerous tissue samples are respectively CC1-CC8, CN1-CN8; and in this embodiment, 8 pairs of gastric cancer tissues and paracancerous tissue samples are respectively GC1-GC8, GN1 -GN8. The TRIZOL extraction method was used to extract the total RNA from the above samples, the absorbance value (A) was measured by an ultraviolet spectrophotometer to determine its content and purity, and the quality of the total RNA was identified by 0.8-1% agarose gel electrophoresis.

[0032] cDNA synthesis: 5 μg of the total RNA extracted from the above-mentioned colon cancer tissue and paired paracancerous tissues and gastric cancer tissues and paired paracancerous tissues w...

Embodiment 2

[0069] Example 2: Clinical Specimen Detection

[0070] (1) 20 pairs of colon cancer tissues and paired paracancerous tissues were taken, and Lnc5q21.2 was amplified by RT-PCR. Template preparation, PCR amplification system and conditions, and detection of amplified products were the same as those in Example 1. Lnc5q21. 2 Relative expression levels in colon cancer paired tissues, the test results are shown in Table 1 below:

[0071] Table 1

[0072]

[0073] (2) Take 20 pairs of gastric cancer tissues and paired paracancerous tissues, and use RT-PCR to amplify Lnc5q21.2. Template preparation, PCR amplification system and conditions, and detection of amplification products are the same as in Example 1, and Lnc5q21.2 is detected. The relative expression levels in gastric cancer paired tissues, the detection results are shown in Table 2 below:

[0074] Table 2

[0075]

Embodiment 3

[0076] Embodiment 3: Sensitivity experiment

[0077] The cDNA samples prepared by the colon cancer cell line HCT116 with high expression of Lnc5q21.2 were selected and ddH 2 O was mixed in proportion, and the above-mentioned Lnc5q21.2 semi-quantitative RT-PCR primer pair was used to detect the expression level of Lnc5q21.2. Amplified products were subjected to agarose gel electrophoresis, such as Figure 5 shown.

[0078] The dilution ratios of the cDNA samples of the colon cancer cell line HCT116 were 100%, 50%, 5%, 1%, 0.5%, and 0%. wxya 2 O is the system control, used to evaluate whether there is contamination of PCR products in the system, such as ddH 2 If the result of O test is negative, the result of the system is credible, and the size of the amplification product of this system is 119bp.

[0079] Figure 5 The results show that: using the specific primer pair, reaction system and conditions for detecting the expression of Lnc5q21.2 in the present invention, the ...

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Abstract

The invention provides a new-found long noncoding RNA Lnc5q21.2, a primer pair for detecting the expression level of the long noncoding RNA in a colon cancer cell line and tissue as well as a kit. The invention firstly finds differential expression of the long noncoding RNA Lnc5q21.2 in colon cancer and para-carcinoma tissues by using a chip technology. A full-length sequence of Lnc5q21.2 is firstly obtained by using a 5'-RACE technology and a 3'-RACE technology. The innovation is shown on the aspect that the expression level of the long noncoding RNA Lnc5q21.2 in colon cancer cell lines, 20 parts of colon cancer tissues and matched para-carcinoma tissues, gastric cancer cell lines as well as 20 parts of gastric cancer tissues and matched para-carcinoma tissues is detected by using specific primers and a semi-quantitative RT-PCR technology. The kit for specifically detecting the expression of the long noncoding RNA Lnc5q21.2 in the colon cancer tissues can be used as a powerful means for colon cancer and gastric cancer diagnosis, curative effect observation, prognosis, residual lesion and recurrence detection and the like; and the kit is simple and convenient to operate, good in stability and high in sensitivity so as to have deep clinical significance and popularization performance.

Description

technical field [0001] The invention relates to a long-chain non-coding RNA and a pair of primers and a kit for detecting the expression level of the long-chain non-coding RNA in cell lines and tissues. Background technique [0002] Colon cancer is one of the most common tumors worldwide. Colorectal cancer is the third most common cancer among men and the second most common among women. In a global cancer statistics in 2008, there were 1.2 million and 608,700 new colon cancer cases and deaths, respectively. Australia, New Zealand, Europe and North America are the high incidence areas. (AhmedinJemal, DVM, Freddie Bray, Melissa M. Center. Global Cancer Statistics. CA CANCER J CLIN 2011.61:69–90) In my country, colon cancer is also one of the common tumors, and its morbidity and mortality are among the top five . (Wanqing Chen, Rongshou Zheng, Siwei Zhang, Ping Zhao. The incidences and mortalities of major cancers in China, 2009. Chin J Cancer. 2013.32 (3): 106-112) In recen...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12Q1/68C12N15/11
Inventor 郭明洲张美英曹宝平高丹令狐恩强
Owner GENERAL HOSPITAL OF PLA
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