Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A.
Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A.
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DOI：
10.1186/1472-6750-6-41
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发表时间：
2006-10-09
影响因子：
3.5
通讯作者：
Peelman LJ
中科院分区：
文献类型：
--
作者：
Erkens T;Van Poucke M;Vandesompele J;Goossens K;Van Zeveren A;Peelman LJ
使用实时RT-PCR的一个重要部分是表达结果必须在得出任何结论之前进行标准化。这可以通过使用一个或多个经验证的参考基因来完成，这取决于结果的所需准确性。然而，在猪中，关于参考基因的表达稳定性的信息非常少。因此，本研究的目的是开发一套新的参考基因，可用于在猪背膘和背最长肌中表达的基因的mRNA表达数据的标准化，这两个代表了猪胴体的经济上重要的部分。过氧化物酶体增殖激活受体γ共激活因子1α（PPARGC 1A）在脂肪代谢和肌纤维类型组成中具有多种功能，是一个非常有趣的肉质候选基因，也是评价我们开发的两种组织类型mRNA表达数据标准化参考基因的理想基因。测定10个参考基因的mRNA表达稳定性。RPL 13 A和SDHA的表达似乎是高度不稳定的。将三个最稳定表达的参考基因（ACTB、TBP和TOP 2B）的几何平均值标准化后，结果不仅显示PPARGC 1A的mRNA表达在每个背最长肌样品中显著高于背膘（P < 0.05），而且在三个肌肉样品中的最头部中的表达也显著高于背膘（P < 0.05）。本研究为猪背膘和背最长肌实时荧光定量PCR数据的标准化提供了一套新的参考基因（ACTB、TBP和TOP 2B）。在应用这组参考基因后，获得的PPARGC 1A表达结果是解开猪中PPARGC 1A表达模式的第一步，并为在保持瘦肉胴体的同时改善肉质的可能选择提供了基础。
An essential part of using real-time RT-PCR is that expression results have to be normalized before any conclusions can be drawn. This can be done by using one or multiple, validated reference genes, depending on the desired accuracy of the results. In the pig however, very little information is available on the expression stability of reference genes. The aim of this study was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in porcine backfat and longissimus dorsi muscle, both representing an economically important part of a pig's carcass. Because of its multiple functions in fat metabolism and muscle fibre type composition, peroxisome proliferative activated receptor γ coactivator 1α (PPARGC1A) is a very interesting candidate gene for meat quality, and was an ideal gene to evaluate our developed set of reference genes for normalization of mRNA expression data of both tissue types. The mRNA expression stability of 10 reference genes was determined. The expression of RPL13A and SDHA appeared to be highly unstable. After normalization to the geometric mean of the three most stably expressed reference genes (ACTB, TBP and TOP2B), the results not only showed that the mRNA expression of PPARGC1A was significantly higher in each of the longissimus dorsi muscle samples than in backfat (P < 0.05), but also that the expression was significantly higher in the most cranial of the three muscle samples (P < 0.05). This study provides a new set of reference genes (ACTB, TBP and TOP2B) suitable for normalization of real-time RT-PCR data of backfat and longissimus dorsi muscle in the pig. The obtained PPARGC1A expression results, after application of this set of reference genes, are a first step in unravelling the PPARGC1A expression pattern in the pig and provide a basis for possible selection towards improved meat quality while maintaining a lean carcass.
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替代剪接和蛋白质功能。
影响因子：
3
作者：
Neverov AD;Artamonova II;Nurtdinov RN;Frishman D;Gelfand MS;Mironov AA
通讯作者：
Mironov AA
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作者：
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DOI：
10.1016/s0301-6226(96)01001-9
发表时间：
1996-12-01
期刊：
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影响因子：
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作者：
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通讯作者：
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影响因子：
2.6
作者：
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通讯作者：
Neuenschwander, S