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Screening method of reference genes in skin tissue of sheep

A technology of internal reference genes and screening methods, which is applied in the field of bioengineering to achieve the effect of improving quantitative accuracy

Inactive Publication Date: 2012-12-26
新疆维吾尔自治区畜牧科学院畜牧科学研究所
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Problems solved by technology

[0005] The invention provides a screening method for an internal reference gene in sheep skin tissue, and the present invention solves the problem that there is no screening method for Chinese Merino sheep (Xinjiang type) GAPDH gene as an internal reference gene in skin tissue

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  • Screening method of reference genes in skin tissue of sheep
  • Screening method of reference genes in skin tissue of sheep
  • Screening method of reference genes in skin tissue of sheep

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Embodiment Construction

[0024] Hereinafter, the present invention will be described in detail with the best embodiment.

[0025] 1 Extraction process:

[0026] The screening method of internal reference genes in sheep skin tissue includes the following steps:

[0027] 1. Method to determine the internal reference gene: take sheep skin tissue as the test sample. First, take 10-20 mg of skin tissue tissue and grind thoroughly in liquid nitrogen. The RNA extraction kit is used to extract total RNA from the skin tissue sample, and 1% agarose gel electrophoresis , 0.5×TBE running buffer, 100V, 15min to detect RNA integrity;

[0028] 2. Take a certain amount of RNA extract, dilute 2-10 times with ddH2O, adjust the spectrophotometer to zero with ddH2O, take the diluent to determine the RNA purity, take the RNA sample with OD260 / OD280 reading between 1.8-2.0 For subsequent experiments, calculate the final concentration of RNA (ng / μL)=(OD260)×(dilution factor 2-10)×40;

[0029] 3. Secondly, take 50ng / μL RNA sample as...

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Abstract

The invention discloses a screening method of reference genes in a skin tissue of sheep, which relates to the technical field of biological engineering. The screening method comprises the steps that: a method of the reference genes is confirmed; a given amount of RNA (ribonucleic acid) extract is taken and diluted by 2-10 times by ddH2O (double-distilled water); a 50 nanograms / microlitre RNA sample is taken to serve as a template, and a cDNA (complementary deoxyribonucleic acid) synthetic reagent kit is adopted for conducting inverse transcription to synthesize cDNA; the cDNA synthesized through the inverse transcription serves as a template, and six reference genes serve as primers; and finally, pairing variation analysis of normalized factors Vn / n+1 of the six reference genes are calculated based on a geNorm program, so as to decide the amount of the optimum reference genes. The screening method solves the problem that no screening method for GAPDH (reduced glyceraldehyde-phosphate dehydrogenase) genes in the skin tissue of a Chinese merino (Xinjiang type) serving as the reference genes is not available at present. Through the combination of the specificity of the SYBR Green I with double chain DNA to generate fluorescence, a real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of the SYBR Green I for the GAPDH genes in the skin tissue of the Chinese merino (Sinkiang type) is established.

Description

Technical field [0001] The present invention relates to the field of bioengineering technology, in particular to a method for screening genes. Background technique [0002] With the release of the complete genome sequence of different species, the quantitative expression level of genes has become increasingly important in the field of life sciences. As an efficient quantitative PCR technology, RT-PCR technology can realize real-time monitoring of a certain gene, and monitor its expression level in different tissues, cells, individuals, and at different developmental stages or after certain special conditions. In order to remove the possible differences in RNA yield, quality, reverse transcription efficiency, and pipettes of different samples to obtain the true difference in the specific expression of the target gene, a specific internal reference gene is usually selected for standardization. Changes in the amplification of internal reference genes can reflect changes in RNA yiel...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 田可川黄锡霞田月珍徐新明狄江哈尼克孜・吐拉甫吴伟伟付雪峰
Owner 新疆维吾尔自治区畜牧科学院畜牧科学研究所
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