Internal Reference Genes, Primers and Applications of Fluorescence Quantitative Quantification of Oyster oyster poly(i:c) Stress Experiment

A technology of real-time fluorescence quantification and internal reference genes, applied in the field of molecular biology, can solve problems such as differences in the expression of housekeeping genes, and achieve the effect of true and reliable data, high accuracy, and universal application

Active Publication Date: 2015-12-09
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

However, under different experimental conditions, the expression of housekeeping genes is still different

Method used

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  • Internal Reference Genes, Primers and Applications of Fluorescence Quantitative Quantification of Oyster oyster poly(i:c) Stress Experiment
  • Internal Reference Genes, Primers and Applications of Fluorescence Quantitative Quantification of Oyster oyster poly(i:c) Stress Experiment
  • Internal Reference Genes, Primers and Applications of Fluorescence Quantitative Quantification of Oyster oyster poly(i:c) Stress Experiment

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Experimental program
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Embodiment 1

[0064] Selection of candidate internal reference genes: We selected five internal reference genes commonly used in long oyster: Elongationfactor-1α, β-actin, 18SrRNA, 28SrRNA and GAPDH, as well as internal reference genes RS18 and RL7 with stable expression during the development of oyster larvae, and another Three homologous genes of human housekeeping genes SKP1, HNRP and UBCD1 obtained from the oyster genome were used as candidate genes for internal reference genes. The selected internal reference gene sequence (see Table 1)

[0065] Experimental materials used in quantitative experiments: adult healthy long oysters (average shell height 90mm) were purchased from Jiaonan, Qingdao. Before the experiment, they were kept in aerated filtered seawater, and the water temperature was maintained at 18°C±1°C. Poly(I:C) stress experiment: 120 long oysters were equally divided into two groups, 60 in each group. In the experimental group, 60 mice were each injected with 100 μl Poly(I...

Embodiment 2

[0068] Evaluate the expression stability of candidate internal reference genes by fluorescent quantitative PCR: design a specific primer pair (see Table 1) according to the candidate gene sequence, perform fluorescent quantitative PCR in Poly(I:C) stress samples, and obtain the expression stability of each candidate gene. The expression data at each stress time point is the Ct value (threshold cycle, the number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value). According to the Ct value, the stable value of each candidate gene was calculated with the GeNorm software commonly used for internal reference screening. The smaller the stable value, the more stable the expression of the gene. In addition, GeNorm software through the pairwise variation coefficient V n / V n+1The analysis can determine the optimal number of housekeeping genes. Vandesompele et al. suggested setting 0.15 as the threshold of the pairwise coefficient ...

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Abstract

The invention discloses fluorescence quantitative reference genes for a crassostrea gigas Poly (I:C) stress experiment as well as primers and applications of the fluorescence quantitative reference genes, relating to the field of molecular biology and particularly relating to the screening of fluorescence quantitative reference genes for a crassostrea gigas polyinosinic-polycytidylic acid stress experiment. The screened beta-actin, GAPDH and RS18 are three reference genes which are most stable in expression in gigas blood cells in the Poly (I:C) stress experiment. The expression stabilities of the three reference genes are superior to the expression stabilities of the other seven reference genes (including common 28S, rRNA and Elongation factor-1alpha) in the Poly (I:C) stress experiment, so that the three reference genes can be simultaneously used as the fluorescence quantitative reference genes to provide powerful support for obtaining precise quantitative data for functional gene expression in the crassostrea gigas Poly (I:C) stress experiment.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an internal reference for functional gene expression analysis in long oyster polyinosinic-polycytidylic acid (Polyinosinic-polycytidylicacid, Poly(I:C)) stress experiments Gene screening. Background technique [0002] The long oyster (Crassostreagigas), also known as the Pacific oyster (Pacificoyster), belongs to the class Bivalvia, the order Pterioida, and the family Oyster. Its meat is tender, delicious, nutritious, and has high economic value. It is an important aquaculture object in the world. Naturally distributed in Japan, China and Korea, it is a worldwide widespread species. Pacific oyster, as a "model species" of shellfish, has been widely valued by scientists, especially the deciphering of the long oyster genome sequence has attracted wider attention recently. [0003] The natural immune system of oysters has long been an area of ​​interest for scientists. The open ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6888C12Q2600/166C12Q2531/113C12Q2545/101
Inventor 黄宝玉张琳琳李莉张国范
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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