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首页> 外文期刊>Journal of Neuroscience Methods >Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.
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Validation of two reference genes for mRNA level studies of murine disease models in neurobiology.

机译:验证两个参考基因在神经生物学中对鼠类疾病模型的mRNA水平研究的有效性。

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摘要

Reverse transcription of extracted cellular RNA combined with real-time PCR is now an established method for sensitive detection and quantification of specific mRNA level changes in experimental models of neurological diseases. To neutralize the impact of experimental error and make quantification more precise, normalization of test gene data using data from a constantly expressed gene, a reference gene that is tested along with the test gene, is required. There is no single gene constantly expressed under all experimental conditions. For a given set of conditions or a given disease model, identification of an unaffected reference gene is necessary. In this report, we present our findings from evaluation and validation of the genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1) and glyceraldehyde phosphate dehydrogenase (GAPDH) as individual reference genes in mRNA level studies involving four murine neurological disease models. We find both genes are suitable as a reference gene with these four models, provided quantification of subtle changes are avoided. We furthermore demonstrate that above a certain threshold of test mRNA level changes and given high quality RNA processing, normalization to total RNA alone provides for equally reliable quantitative mRNA level results.
机译:提取的细胞RNA的逆转录与实时PCR的结合现已成为一种灵敏检测和定量神经疾病实验模型中特定mRNA水平变化的方法。为了抵消实验误差的影响并使定量更加精确,需要使用来自恒定表达基因的数据对测试基因数据进行标准化,该基因是与测试基因一起被测试的参考基因。在所有实验条件下都没有一个恒定表达的基因。对于给定的一组条件或给定的疾病模型,必须确定未受影响的参考基因。在本报告中,我们从评估和验证编码次黄嘌呤鸟嘌呤磷酸核糖基转移酶1(HPRT1)和甘油醛磷酸脱氢酶(GAPDH)的基因作为涉及四个鼠类神经疾病模型的mRNA水平研究中的单个参考基因中提出了我们的发现。我们发现这两个基因都适合作为这四个模型的参考基因,但要避免量化细微的变化。我们进一步证明,在测试mRNA水平变化的某个阈值之上并考虑到高质量的RNA处理,单独对总RNA的标准化可提供同样可靠的定量mRNA水平结果。

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