Dual-gene micro RNA fluorescent quantitative internal reference and application of primer in shellfish development samples

A fluorescent quantitative and dual-gene 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 simple operation

Active Publication Date: 2014-04-09
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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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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  • Dual-gene micro RNA fluorescent quantitative internal reference and application of primer in shellfish development samples
  • Dual-gene micro RNA fluorescent quantitative internal reference and application of primer in shellfish development samples
  • Dual-gene micro RNA fluorescent quantitative internal reference and application of primer in shellfish development samples

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Embodiment 1

[0043] Selection of candidate internal reference genes: We performed microRNA sequencing on samples of Oyster oyster at different developmental stages, and obtained some newly identified microRNAs and their expression profiles. Based on the miRNA expression profiling data, we selected 6 microRNAs with relatively stable expression as candidate genes (ie, miR-8, miR-12, miR-29, miR-71, miR-92, miR-1992). 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, Hu W, Yan Q, et al. (2009) Genome-wide identification of Schistosoma japonicum microRNAs using a deep...

Embodiment 2

[0049] 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 developmental samples, and obtain the expression data of each candidate gene at each developmental stage—Ct value (threshold cycle, The number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value). After converting the Ct value data with formula 2-Ct, 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.2004Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify ge...

Embodiment 3

[0068] The two-gene combination is used as an internal reference, and the expression Ct value of the two-gene internal reference is the arithmetic mean of the expression Ct values ​​of miR-71 and miR-8 genes in a certain developmental sample. Using the method based on SYBR Green dye fluorescence quantitative PCR, the relative quantitative analysis of two randomly selected long oyster miRNAs (miR-9 and m0315) in 13 developmental samples was carried out, see the literature: 【Shi R, Chiang VL(2005) Facile means for quantifying microRNA expression by real-time PCR.Biotechniques255039:519-525.]. 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.

[0069] Relative quantification of long oyster miRNA:

[0070]Obtaining of developmental samples, RNA extraction and synthes...

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Abstract

The invention relates to the field of molecular biology, in particular to a dual-gene micro RNA (miRNA) fluorescent quantitative internal reference and application of a primer in shellfish development samples. The two genes (miR-71 and miR-8) are selected by the strict internal reference screening procedure and are the most stable miRNA genes in different development stages of shellfish miRNA, the expression stability of the two genes in different development stages is superior to that of common miRNA internal reference genes snRNA U6, snRNA U1 and 5.8S RNA. Two miRNAs are selected randomly, the dual-gene miRNA is used as the internal reference for carrying out fluorescent quantitative analysis in development samples, the expression results are highly accordance with the miRNA sequencing expression profile, and the reliability and the stability of the dual-gene internal reference are proved.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a dual-gene miRNA quantitative internal reference and primers suitable for microRNA (miRNA) expression analysis in shellfish developmental samples. Background technique [0002] Shellfish, belonging to the clambranch (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 h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
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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