Optimization of ribonucleic acid detection from archival Guinea pig temporal bone specimens.
Optimization of ribonucleic acid detection from archival Guinea pig temporal bone specimens.
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档案豚鼠颞骨标本核糖核酸检测的优化。
DOI：
10.1097/01.mao.0000235377.70492.c7
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发表时间：
2007
期刊：
影响因子：
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通讯作者：
Megerian,CliffA
中科院分区：
文献类型：
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作者：
Hall,KathrynL;Pitts,DarrellR;Anne,Samantha;Semaan,MarounT;Alagramam,KumarN;Megerian,CliffA
背景：大量的档案颞骨存在。利用RNA提取技术对这些标本进行回顾性分析将极大地丰富我们对特定耳科疾病病理生理学的理解。然而，档案人类颞骨老化并包埋在石蜡或火棉胶中，使得在保存的标本中分离和操作核酸变得困难，特别是因为它涉及RNA降解。尽管最近有一些适度成功的报道，但使用聚合酶链反应（PCR）分析的RNA分离和基因表达仍然具有挑战性和不可靠性。本研究利用豚鼠颞骨标本建立了RNA提取和PCR及定量PCR技术分析基因表达的方法。扩增的基因包括管家基因和与谷氨酸pathway.Methods相关的基因：从资深作者收集的实验性水肿内耳标本中收集档案纤维素包埋的豚鼠颞骨。使用先前描述的方案在16只动物中提取来自该组织的RNA，并使用改良的trizol提取技术在10只动物中提取RNA。对提取的RNA进行基因表达分析。分析包括两个管家基因，GAPDH和18 S，以及三个介质的谷氨酸途径，谷氨酸天冬氨酸转运蛋白，谷氨酸合成酶，诱导型一氧化氮synthes.Results：与标准提取方案相比，三唑为基础的提取技术表现出更高的可靠性和重复性的RNA检测。用标准方法检测的36次尝试中有7次检测到管家基因GAPDH或18 S，而用改进的提取方法检测的9次尝试中有9次检测到管家基因GAPDH或18 S（P< 0.001）。目标基因谷氨酸-天冬氨酸转运蛋白在标准方案的26次尝试中有3次检测到，而在改良提取方法的13次尝试中有12次检测到（P< 0.001）。信使RNA水平的定量，然后实现使用定量PCR methods.Conclusion：改进的可靠性检测基因表达和示范的重现性完成了修改的RNA提取技术和标准的逆转录酶PCR协议。此外，我们还表明，档案材料的基因表达可以通过实时PCR定量。
Background:A large number of archival temporal bones exist. Retrospective analysis of these specimens using techniques of RNA extraction will greatly enrich our understanding of the pathophysiology of specific otologic diseases. However, archival human temporal bones are aged and embedded in paraffin or celloidin, rendering isolation and manipulation of nucleic acid in preserved specimens difficult, especially as it pertains to RNA degradation. Despite some reports of moderate success in the recent past, RNA isolation and gene expression using polymerase chain reaction (PCR) analysis continues to be challenging and unreliable. Archival guinea pig temporal bone specimens were used to develop and optimize a protocol for RNA extraction and gene expression analysis using PCR and quantitative PCR methods. The genes amplified comprise housekeeping genes and genes associated with the glutamate pathway.Methods:Archival celloidin-embedded guinea pig temporal bones were collected from the senior author's collection of experimental hydropic inner ear specimens. RNA from this tissue was extracted using the protocol described previously in 16animals and using a modified trizol extraction technique in 10 animals. Gene expression analysis was performed on the extracted RNA. Analysis included two housekeeping genes, GAPDH and 18S, as well as three mediators of the glutamate pathway, glutamate aspartate transporter, glutamate synthetase, and inducible nitric oxide synthase.Results:Compared with the standard extraction protocol, the trizol-based extraction technique showed greater reliability and reproducibility of RNA detection. The housekeeping gene GAPDH or 18S was detected in 7 of 36 attempts with the standard protocol versus 9 of 9 using the modified extraction method (P< 0.001). The gene of interest, glutamate aspartate transporter, was detected in 3 of 26 attempts with the standard protocol versus 12 of 13 attempts using the modified extraction method (P< 0.001). Quantification of messenger RNA levels was then achieved using quantitative PCR methods.Conclusion:Improved reliability for detection of gene expression and demonstration of reproducibility were accomplished by modification of RNA extraction technique and standard reverse transcriptase PCR protocol. In addition, we also showed that gene expression from archival material can be quantified by real-time PCR.