Quantification of insulin-like growth factor-1 (IGF-1) mRNA: Development and validation of an internally standardised competitive reverse transcription-polymerase chain reaction
Quantification of insulin-like growth factor-1 (IGF-1) mRNA: Development and validation of an internally standardised competitive reverse transcription-polymerase chain reaction
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DOI：
10.1055/s-0029-1212025
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发表时间：
1998-01-01
影响因子：
1.8
通讯作者：
Sauerwein, H
中科院分区：
文献类型：
--
作者：
Pfaffl, M;Meyer, HHD;Sauerwein, H
为了研究各种组织中局部IGF-1 mRNA表达的作用，我们开发并验证了一种允许对IGF-1 mRNA进行特异性、灵敏性和可靠性定量的方法：内部标准化逆转录聚合酶链反应（RT-PCR）。设计了合成的竞争性模板IGF-1标准cRNA（IGF-1 cRNA），其含有用于扩增野生型IGF-1 mRNA的相同侧翼引物序列，但长度相差56 bp。为了获得组织RNA样品中存在的IGF-1 mRNA浓度，将一系列250 ng总RNA掺入三种已知量的标准IGF-1 cRNA，孵育用于竞争性RT-PCR反应，随后分离获得的两种扩增产物（184 bp来自IGF-1 cRNA，240 bp来自野生型IGF-1 mRNA），并通过HPLC-UV定量。对于每个单独的组织RNA样品，将比率R（R =竞争者PCR产物/野生型PCR产物）相对于竞争者IGF-1 cRNA的Starling分子的数量作图。然后可以读出样品中存在的IGF-1 mRNA的初始量，其中R = 1。经验证的试验的检测限为1600个IGF-1 cRNA分子/反应，试验内变异为7.4%（n = 5），在140 ng至840 ng总RNA输入之间具有线性（r = 0.997）。本方法首次应用于研究长期去势对牛组织中IGF-1表达率的影响。肝脏IGF-1 mRNA浓度与RIA定量的血浆浓度具有良好的相关性（r = 0.81），并且完整动物中IGF-1 mRNA浓度高于去势动物。在两个骨骼肌（m。夹肌和肌腓肠肌）IGF-1 mRNA浓度分别比肝脏低20倍和35倍，在阉牛和公牛之间没有任何差异。在公牛中，IGF-1 mRNA表达量在雄性中较高。夹肌（p < 0.01）较M.腓肠肌，表明本地产生的IGF-1可能是重要的性二型肌肉，生长模式。
To investigate the role of local IGF-1 mRNA expression in various tissues, we developed and validated a method which allows for a specific, sensitive and reliable quantification of IGF-1 mRNA: an internally standardised Reverse Transcription-Polymerase Chain Reaction (RT-PCR). A synthetic competitive template IGF-1 standard cRNA (IGF-1 cRNA) was designed, which contains the same flanking primer sequences used to amplify the wild type IGF-1 mRNA, but differs by 56 bp in length. To obtain the IGF-1 mRNA concentration present in tissue RNA samples, series of 250 ng total-RNA were spiked with three known quantities of the standard IGF-1 cRNA, incubated for competitive RT-PCR reactions and the two amplificates obtained (184 bp from IGF-1 cRNA and 240 bp from the wild type IGF-1 mRNA) were subsequently separated and quantified by HPLC-UV. For every individual tissue RNA sample, the ratio R (R = competitor PCR product / wild type PCR product) was plotted against the number of starling molecules of the competitor IGF-1 cRNA. The initial amount of IGF-1 mRNA present in the sample can then be read off where R = 1. The validated assay had a detection limit of 1600 IGF-1 cRNA molecules/reaction, the intra-assay variation was 7.4% (n = 5) and linearity (r = 0.997) was given between 140 ng to 840 ng total-RNA input. The present method was first applied to study the effect of long: term castration on the IGF-1 expression rates in bovine tissues. The hepatic IGF-1 mRNA concentrations were well correlated (r = 0.81) with the plasma concentrations as quantified by RIA and were higher in intact than in castrated animals. In two skeletal muscles (m. splenius and m. gastrocnemius) IGF-1 mRNA concentrations were 20- and 35- times lower than in liver, respectively, without any differences between steers and bulls. In bulls, the IGF-1 mRNA expression was higher in m. splenius (p < 0.01) than m. gastrocnemius, indicating that locally produced IGF-1 might be important for sexually dimorphic muscle,growth patterns.