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Internal reference gene hsa_circ_0000471 of human tissue/cell circular RNA and its application

A technology of human tissue and internal reference genes, applied in the field of molecular biology, can solve problems such as the inability to rule out the interference of linear shear isomers and correct the expression of target genes

Active Publication Date: 2021-03-30
JIANGSU CANCER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the back-to-back primers can exclude the interference of the parental linear gene to a certain extent, the interference of the linear splice isoform with the same back-splice junction sequence as the circular RNA cannot be excluded
The best practice is to use RNase R to remove the interference of these linear splicing isoforms, but this will also remove β-actin or GAPDH as internal reference genes, so these linear internal reference genes can no longer be used to correct the expression of the target gene

Method used

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  • Internal reference gene hsa_circ_0000471 of human tissue/cell circular RNA and its application
  • Internal reference gene hsa_circ_0000471 of human tissue/cell circular RNA and its application
  • Internal reference gene hsa_circ_0000471 of human tissue/cell circular RNA and its application

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Experimental program
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Effect test

Embodiment 1

[0017] 1. Screen candidate internal reference genes for circular RNA

[0018] Log in to the Gene Expression Omnibus (GEO) website (https: / / www.ncbi.nlm.nih.gov / geo / ) to download the dataset numbered GSE77661 (GSE77661_circ_srpbm_union_samples_loci.txt.gz), which includes 6 healthy human specimens (brain, colon, heart, liver, lung, and stomach) and 7 pairs of cancerous and paracancerous human tissue specimens (bladder, breast, colorectal, gastric, liver, clear cell, and prostate) circRNA-sequencing result.

[0019] The data processing method is as follows:

[0020] (1) Only the circular RNAs expressed in all samples were retained, and a total of 50 circular RNAs were obtained.

[0021] (2) Analyze the stability of the 50 circular RNAs obtained in tissue samples using geNorm, NormFinder and BestKeeper respectively, take the 10 circular RNAs with the best stability in each method, and then combine the obtained 3 data Set the intersection and get 6 circular RNAs.

[0022] 2. F...

Embodiment 2

[0038] Evaluation of specificity and amplification efficiency of hsa_circ_0000471

[0039] The results of RT-qPCR showed that the melting curve of hsa_circ_0000471 was a single peak ( Figure 1a shown), indicating that there is no non-specific amplification. In order to further confirm that there is no amplification of non-specific bands, 2% agarose gel electrophoresis was used to amplify the product, and the results were as follows: Figure 1b As shown, no miscellaneous bands were seen.

[0040] In order to obtain the amplification efficiency of hsa_circ_0000471, the template was first diluted, and then amplified on the machine. After the amplification result was obtained, the result was fitted with a straight line to obtain the slope (slope), and then the amplification efficiency was calculated using the following formula:

[0041] E(%)=(2 -1 / slope -1)×100

[0042] Finally, the amplification efficiency of hsa_circ_0000471 was found to be 99.93%.

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Abstract

The invention discloses a reference gene has_circ_0000471 of fluorogenic quantitative PCR (RT-qPCR) of human tissue / cell specimen circRNA, a primer and an application thereof. According to the invention, a high-throughput sequencing result is firstly screened, and then RT-qPCR is utilized to verify in various cells of human body; a result proves that the acquired reference gene and the specific primer thereof used for analyzing relative expression of human body are accurate and stable; the expression level is not influenced by experimental conditions, such as chemotherapeutics and anoxia.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to an RT-qPCR internal reference gene hsa_circ_0000471 of human tissue / cell specimen circular RNA and its primer and application. Background technique [0002] Circular RNAs (circular RNAs, circRNAs) are a class of molecules with a single-stranded closed circular structure formed by covalent bonds. There is no 5' end cap and 3' end poly(A) tail, and the structure is relatively stable and is not affected by ribonucleases. Effect of R (RNase R). Humans have discovered the existence of circular RNA molecules in 1976, but they have been regarded as faulty splicing products or mediators that escape intron lariat debranching. In recent years, with the development of high-throughput sequencing technology and the update of analysis methods, a large number of circular RNA molecules have been identified. More and more studies have shown that circular RNA molecules are ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/6851C12N15/11
CPCC12N15/113C12N2310/532C12Q1/6851C12Q2531/113C12Q2545/101
Inventor 冯继锋钟山亮周思颖杨苏晋俞心念林振中
Owner JIANGSU CANCER HOSPITAL
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