Large White, an introduced European pig breed, and Meishan, a Chinese indigenous pig breed, were hybridized directly and reciprocally and a total of 260 pigs, including purebreds, Large White and Meishan, and their hybrids, White${\times}$Meishan (LM) and Meishan${\times}$Large...
Large White, an introduced European pig breed, and Meishan, a Chinese indigenous pig breed, were hybridized directly and reciprocally and a total of 260 pigs, including purebreds, Large White and Meishan, and their hybrids, White${\times}$Meishan (LM) and Meishan${\times}$Large White (ML) pigs, were bred in our laboratory. The mRNA differential display PCR (DD-PCR) was used to detect the age-dependent changes of differential gene expression in backfat tissue between hybrids and parents. Some measures were taken to reduce the false positives in our experiment. Among the total of 2,686 bands obtained, 1,952 bands (about 72.67%) were reproducible and eight patterns (fifteen kinds) of gene expression were observed. The percentage of differentially expressed genes between hybrids and parents is 56.86% at the age of four months and 57.71% at the age of six months. This indicated that the differences of gene expression between hybrids and their parents were very obvious. U-test was used to compare the patterns of gene expression between the age of four and six months, and results showed that bands occurring in only one hybrid and bands displayed in one hybrid and one parent were significantly different at p<0.05, and bands visualized in only two hybrids were significantly different at p<0.01. These indicated that differential gene expression between hybrids and parents changed at different ages.
Large White, an introduced European pig breed, and Meishan, a Chinese indigenous pig breed, were hybridized directly and reciprocally and a total of 260 pigs, including purebreds, Large White and Meishan, and their hybrids, White${\times}$Meishan (LM) and Meishan${\times}$Large White (ML) pigs, were bred in our laboratory. The mRNA differential display PCR (DD-PCR) was used to detect the age-dependent changes of differential gene expression in backfat tissue between hybrids and parents. Some measures were taken to reduce the false positives in our experiment. Among the total of 2,686 bands obtained, 1,952 bands (about 72.67%) were reproducible and eight patterns (fifteen kinds) of gene expression were observed. The percentage of differentially expressed genes between hybrids and parents is 56.86% at the age of four months and 57.71% at the age of six months. This indicated that the differences of gene expression between hybrids and their parents were very obvious. U-test was used to compare the patterns of gene expression between the age of four and six months, and results showed that bands occurring in only one hybrid and bands displayed in one hybrid and one parent were significantly different at p<0.05, and bands visualized in only two hybrids were significantly different at p<0.01. These indicated that differential gene expression between hybrids and parents changed at different ages.
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제안 방법
This technique possesses the advantages over other similar techniques as follows: more than two samples can be compared simultaneously, and only a small amount of total RNA is needed. In the present experiment, the aim is to detected the difference of gene expression of backfat tissue at the age of four and six months between purebreds, Large White and Meishan, and their hybrids, WhitexMeishan (LM) and MeishanxLarge White (ML) pigs.
The PCR was done as follows: 94°C for 5 min, 40°C for 5 min, 72°C for 5 min, 3 cycles of 94°C for 2 min, 40°C for 2 min, 72°C for 5 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, and then 72°C extension for 10 min.
5 μl sterile water. The PCR was done as follows: 94°C for 5 min, 40°C for 5 min, 72°C for 5 min, 3 cycles of 94°C for 2 min, 40°C for 2 min, 72°C for 5 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, and then 72°C extension for 10 min.
The key problems of this technique were the reproducibility of display PCR and the high percentage of false positive. The display PCR conditions were optimized to minimally reduce false positive (Liang et al.
대상 데이터
Ten 5’ arbitrary primers in combination with ten 3’anchor primers (100 sets) were used in this experiment.
But the use of long differential display primers and the integration of high and low annealing temperature must lose some information of differentially expressed mRNA, resulting in statistically inaccuracy. Ten 5’ arbitrary primers in combination with ten 3’anchor primers (100 sets) were used in this experiment. All the patterns of gene differential expression between hybrids and parents were examined and the information of differentially expressed mRNA was farthest embodied.
The following components were added to the annealed primer/template: 5 μl of M-MLV 5xReaction Buffer, 1.25 μl of 10 mM dNTPs, 25 units of RNasin® Ribonuclease Inhibitor (Promega, USA), 200 units of MMLV RT (Promega, USA), nuclease-free ddH2O to a final volume of 25 μl and mixed gently by flicking the tube.
성능/효과
, 2004). In this experiment, the measures were taken to reduce false positive and the result showed that 72.67% bands could be reproducible. The percentage of bands reproducible increased about 4% more than that reported by Xie (2003) and about 6% reported by Wang (2004).
The percentage of each pattern was different between the age of four and six months, respectively, and U-test of the patterns of gene expression between the age of four and six months showed that bands occurring in only one hybrid and bands occurring in one hybrid and one parent were significantly different at p<0.05, and bands visualized in only two hybrids was significantly different at p<0.01 (Table 1).
후속연구
These patterns might have a different influence on the performance of trait at different age. However, this work was preliminary, and more research should be performed to isolate, functionally analyze the differentially expressed genes and make clear the reasons leading to gene differential expression, consequently, go deep into understanding age-dependant changes of differential gene expression between hybrids and parents.
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