Reference genes stably expressed in different tissues of pigs, and screening method and application thereof

An internal reference gene and stable expression technology, applied in the field of bioengineering, to achieve the effect of excellent expression stability

Inactive Publication Date: 2019-05-24
FOSHAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many factors, such as: RNA quality, purity, different sample sizes, enzyme activity, and PCR amplification efficiency will affect the results of RT-qPCR detection of gene expression

Method used

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  • Reference genes stably expressed in different tissues of pigs, and screening method and application thereof
  • Reference genes stably expressed in different tissues of pigs, and screening method and application thereof
  • Reference genes stably expressed in different tissues of pigs, and screening method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1 RT-qPCR of internal reference genes in different tissues of pigs

[0023] 1. Selection of reference genes in different tissues of pigs and design of primers

[0024] The present disclosure selects 9 candidate genes according to different tissues of pigs, namely Top2b, Actb, Rps18, Ppia, 18S, Tbp, Hmbs, B2m and Rpl4. Based on the relevant gene sequences published by NCBI, 9 pairs of specific primers were designed and synthesized. The sequences are shown in Table 1:

[0025] Table 1 Primer sequence list

[0026]

[0027] 2. Extraction of total RNA from different tissues of pigs and synthesis of cDNA

[0028] Seven different porcine tissues (heart, liver, spleen, lung, kidney, muscle, subcutaneous fat) were collected from three 15-day-old Bama miniature pigs. All samples were immediately snap-frozen in liquid nitrogen and stored at −80 °C prior to extraction of total RNA.

[0029] Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA)...

Embodiment 2

[0033] Example 2 Stability Analysis of Internal Reference Gene Expression in Different Tissues of Pigs

[0034] For the amplification results obtained in Example 1 above, use the relative expression level Q=2 －ΔΔCt (ΔCt = sample Ct value - minimum Ct value) method to calculate the relative expression level of the target gene, which was imported into GeNorm and NormFinder for calculation. The GeNorm program screens out internal references with better stability by calculating the M value of the stability of each internal reference gene. The judgment standard is that the smaller the M value, the better the stability of the internal reference gene, and vice versa, the worse the stability. The software can also Calculate the paired variation V value of the normalization factor after introducing a new internal reference gene, and determine the most appropriate internal parameter value according to the Vn / Vn+1 value. NormFinder can also calculate the stable value of internal referen...

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Abstract

The invention provides reference genes stably expressed in different tissues of pigs. The reference genes are Rps18, Rpl4, Ppia and Tbp, which have excellent expression stability in the different tissues of the pigs, providing a selection basis for studying the level of gene expression in the different tissues of the pigs.

Description

technical field [0001] The disclosure relates to the technical field of bioengineering, in particular to an internal reference gene stably expressed in different tissues of pigs, a screening method and application thereof. Background technique [0002] Pigs (sus scrofa) are an economically important livestock in meat production. Bama miniature pigs are one of the main miniature pig breeds in China. Due to their good genetic stability, good reproductive performance, light weight, large white hair coverage on the body surface, multiple tissues and organs and biochemical indicators similar to humans, it has also become a Ideal animal models for studying human disease. Therefore, in the study of improving meat quality and human diseases, real-time fluorescent quantitative PCR is used to quantitatively analyze the target genes in Bama miniature pigs. Accurate gene expression patterns are of great significance for gene function analysis. [0003] Among them, real-time fluorescen...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12Q1/6888C12N15/11C12Q1/6806
Inventor裴杨莉李奎李华
OwnerFOSHAN UNIVERSITY