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首页> 外文期刊>Brain research. Brain research protocols >Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.
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Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

机译：快速PCR介导的竞争分子的合成，用于准确定量β（2）GABA（A）受体亚基mRNA。

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摘要

We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.
机译：我们描述了一种基于PCR的非特异性条件合成竞争对手分子的快速简便的方法。 RT竞争性PCR是一种敏感的技术，可以对小组织样品中非常少量的mRNA分子进行定量。该技术基于PCR期间天然和标准模板之间针对核苷酸，引物或其他因素的竞争。因此，最关键的参数是使用良好的内标生成标准曲线，从中可以正确估计天然序列的数量。目前，已经描述了不同类型的内标及其合成方法。通常，这些方法大多数都是耗时的，需要使用不同的引物组，不同轮次的PCR或特定的修饰，例如定点诱变，需要对PCR产物进行后续分析。使用我们的方法，我们在单轮PCR中使用相同的引物对，获得了成功用于RT竞争性PCR实验的竞争分子。这种方法的主要优点是通用性强且经济。从理论上讲，有可能为每个使用的引物对合成一个特定的竞争分子。最后，使用这种方法，我们已经能够量化在大鼠海马发育过程中发生的beta（2）GABA（A）受体亚基mRNA表达的增加。

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