Detection method and primers for evaluating enrichment result of porcine histone modification
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A detection method and histone technology, applied in the field of ChIP-seq, can solve the problems of high cost of ChIP-seq, poor stability between materials, few applications, etc., and achieve the effect of saving time and economic cost
Active Publication Date: 2020-12-01
WUHAN IGENEBOOK BIOTECH CO LTD
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[0005] However, the main materials of ChIP technology research are concentrated on cell or tissue samples of model animals, and the processing methods are relatively mature, but the application in porcine muscle and adipose tissue is less, especially adipose tissue has high requirements for pre-treatment and is difficult to break. It is difficult to extract complete nuc...
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Embodiment 1
[0067] Example 1, quantitative detection of H3K4ME3 and H3K27AC enrichment in porcine adipose tissue.
[0068] In this embodiment, pig adipose tissue is used as a sample, and its specific operation steps are as follows,
[0069] 1) First take a 50ml centrifuge tube, add 50ml PBS 100ul PIS 200ul PMSF to pre-cool on ice, and then sample 0.5g of muscle tissue.
[0070] 2) Cut the sample into small pieces with scissors, add PBS to make it 30ml, then add 840ul 37% formaldehyde, and shake gently on a shaker at room temperature for 15min.
[0071] 3) Add 3.42ml of 2.0M glycine to make the final concentration 0.2M, shake gently on a shaker at room temperature for 5min.
[0072] 4) Centrifuge at room temperature for 5 minutes at 2500 rpm.
[0073] 5) The lower layer was washed twice with PBS, and centrifuged at 2500 rpm at 4° C. for 5 min.
[0074] 6) The liquid was discarded, and the precipitate was homogenized with cell lysate; wherein, the amount of cell lysate added was 3 mL of ...
Embodiment 2
[0092] Example 2, quantitative detection of H3K4ME3 and H3K27AC enrichment in porcine muscle tissue.
[0093] In this embodiment, pig muscle tissue is used as a sample, and the specific operation steps are the same as those in Embodiment 1.
[0094] The result is as figure 2 shown.
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Abstract
The invention provides a detection method and primers for evaluating an enrichment result of porcine histone modification. The method comprises the following steps: enriching DNA combined with targetprotein by using a pig tissue sample through a ChIP-seq method, carrying out quantitative detection by adopting a fluorescent quantitative QPCR method, and judging the result, wherein a calculation method of the quantitative detection result adopts a delta delta Ct method for judgment, and the result is judged as follows: if the effective precipitation enrichment multiple of positive primers and negative primers is greater than or equal to 2, the enrichment effect is considered to reach the standard, and if the effective precipitation enrichment multiple of positive primers and negative primers is less than 2, the enrichment effect is considered to not reach the standard. According to the method, the v histone modification level can be specifically detected, and the DNA region enrichment effect can be effectively judged, so that important reference is provided for ChIP-seq, the time and economic cost caused by poor sequencing result due to poor enrichment effect are greatly saved, andan effective way is provided for developing pig epigenomics research.
Description
technical field [0001] The invention relates to the technical field of ChIP-seq, in particular to a detection method and primers for evaluating the enrichment result of porcine histone modification. Background technique [0002] Fat deposition and muscle growth are one of the most important economic traits of pigs, which directly affect the quality of pork and the level of its economic value. Therefore, it is a very important subject to study the genetic traits of pig fat and muscle tissue through genetics. [0003] Among them, epigenetics research is a newly developed scientific field after genetics, which is to study the heritable expression of genes without changing the nucleotide sequence of genes. Histone modification is an important regulatory mode of epigenetic regulation, which can regulate gene expression through methylation and acetylation modification. H3K4ME3 and H3K27ac are important epigenetic modifications of histone modification, which have been expressed i...
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