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Application of a dual-gene microRNA fluorescence quantitative internal reference and its primers in shellfish tissue samples

A fluorescence quantitative and tissue sample technology, applied in the field of molecular biology, can solve the problems of high cost and unsuitability for miRNA function analysis, and achieve the effect of low cost, high sensitivity and improved accuracy

Active Publication Date: 2015-09-23
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Large-scale sequencing and gene chip methods are high-throughput methods, which are expensive and not suitable for functional analysis of a small number of miRNAs

Method used

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  • Application of a dual-gene microRNA fluorescence quantitative internal reference and its primers in shellfish tissue samples
  • Application of a dual-gene microRNA fluorescence quantitative internal reference and its primers in shellfish tissue samples
  • Application of a dual-gene microRNA fluorescence quantitative internal reference and its primers in shellfish tissue samples

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Selection of candidate internal reference genes: We performed microRNA sequencing on different tissue samples of oyster oyster, and obtained some newly identified microRNAs and their expression profiles. Based on the miRNA expression profiling data, we selected five microRNAs with relatively stable expression as candidate genes (ie, let-7, miR-87, miR-1994, miR-71, miR-1). At the same time, the commonly used microRNA internal reference gene 5.8S RNA, snRNA U6 and snRNA U1 were used as candidate genes.

[0044] The miRNA sequence (see Table 1) and sequencing expression profile of Oyster oyster are the results given by the RNA samples provided by us and sequenced and analyzed by Shenzhen BGI Institute. The following is a brief introduction to the method of deep sequencing analysis. For details, please refer to the literature [Huang J, Hao P, Chen H, HuW, Yan Q, et al. (2009) Genome-wide identification of Schistosoma japonicum microRNAs using a deep-sequencing approach. P...

Embodiment 2

[0048] Evaluate the expression stability of candidate internal reference genes by fluorescent quantitative PCR: design specific primers according to the candidate gene sequence, perform fluorescent quantitative PCR in tissue samples, and obtain the expression data of each candidate gene in each tissue—Ct value (threshold cycle, per The number of cycles experienced when the fluorescent signal in a reaction tube reaches the set threshold value). Use the Ct value data with Equation 2 -Ct After conversion, use the most commonly used excel algorithm NormFinder for internal reference screening to calculate the stable value of each candidate gene. The smaller the stable value, the more stable the gene expression. For the specific algorithm and principle of NormFinder, please refer to the literature [ANDERSEN, C.L., JENSEN, J.L.&ORNTOFT, T.F.2004 Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited ...

Embodiment 3

[0067] The double-gene combination is used as an internal reference, and the expression level Ct value of the double-gene internal control is the arithmetic mean of the expression level Ct values ​​of the let-7 and miR-87 genes in a certain tissue sample. Using the method based on SYBR Green dye-based fluorescent quantitative PCR, the relative quantitative analysis of two randomly selected long oyster miRNAs (miR-7 and m0078) in four tissues (mantle, adductor muscle, gills, and lip flaps) was carried out. See literature: [Shi R, Chiang VL (2005) Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 2550 39: 519-525.]. It was found that the results of fluorescent quantitative PCR using the double gene as an internal reference were highly consistent with the results of the sequencing expression profile, which proved the reliability and stability of the dual gene internal reference.

[0068] Relative quantification of long oyster miRNA:

[0069] Obtain...

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Abstract

The invention relates to the field of biology, and particularly relates to application of dual-genetic microRNA (miRNA) fluorescent quantitative internal references and a primer thereof in shellfish tissue samples. Two genes (let-7 and miR-87) selected by the invention are selected according a strict internal reference screening process, and are miRNA genes with the most stable expression in the shellfish microRNA in different tissues; the expression stability of the genes in the tissue is superior to the common miRNA internal reference genes snRNA U6, snRNA U1 and 5.8S RNA. Two miRNAs are selected at random, and the dual-genetic miRNAs are taken as internal references to carry out fluorescence quantitative analysis in the tissue samples; the obtained expression result is highly identical to miRNA sequencing expression profile; the reliability and the stability of the dual genetic internal reference are proved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a double-gene miRNA quantitative internal reference and primers suitable for microRNA (miRNA) expression analysis in shellfish tissue samples. Background technique [0002] Shellfish, belonging to the clambranch class (or bivalves) in the mollusk phylum, common species include oysters, mussels, clams, and razor clams. There are about 10,000 species in existence, 80% of which live in the ocean. Most species of shellfish are edible. Many shellfish are tender, delicious, nutritious and have high economic value. They are important aquaculture objects in the world. [0003] MicroRNA is a kind of small molecule non-coding single-stranded RNA that widely exists in the biological world. It is about 21-24nt long. It is widely expressed in different tissues and organs and participates in various physiological activities of organisms. Because of its importance in organisms, scientists at ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/158C12Q2600/166C12Q2600/178
Inventor 黄雯阙华勇许飞李莉张国范
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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