Combining laser capture microdissection with quantitative real-time PCR: Effects of tissue manipulation on RNA quality and gene expression
Combining laser capture microdissection with quantitative real-time PCR: Effects of tissue manipulation on RNA quality and gene expression
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DOI：
10.1016/j.jneumeth.2005.10.010
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发表时间：
2006-05-15
影响因子：
3
通讯作者：
Watson, Stanley J.
中科院分区：
文献类型：
--
作者：
Kerman, Ilan A.;Buck, Bradley J.;Watson, Stanley J.
激光捕获显微切割（LCM）越来越多地用于神经系统的定量基因表达研究。本研究旨在确定LCM研究中各种组织操作对提取RNA完整性的影响。我们的数据表明，显微切割前的各种组织制备策略可能会降低RNA质量高达25%，从而影响某些基因的表达谱。为了解决这个问题，我们开发了一种逆转录酶实时PCR策略，该策略具有相当高的灵敏度，可用于计算基因表达的相对变化。该方法在大鼠小脑的亚区域中得到验证。因此，神经胶质基因标记物髓鞘相关糖蛋白和蛋白脂质蛋白1的表达在小脑皮质的白色物质层中比在神经元富集的颗粒层中高70-160倍。相比之下，与白色物质层相比，颗粒层中特定神经元标记物（神经元特异性烯醇化酶）的表达高出七倍。此外，这种方法具有较高的灵敏度和特异性，因为我们能够检测到神经元特异性烯醇化酶的表达降低38%，而神经胶质标记物的表达在给予神经毒素鹅膏蕈氨酸后没有变化。这些结果证明了在LCM样品中进行准确的半定量基因表达分析的可行性。(c)2005 Elsevier B. V.保留所有权利。
Laser capture microdissection (LCM) is increasingly being used in quantitative gene expression studies of the nervous system. The current study aimed at determining the impact of various tissue manipulations on the integrity of extracted RNA in LCM studies. Our data indicate that various tissue preparation strategies prior to microdissection may decrease RNA quality by as much as 25%, thus affecting expression profiles of some genes. To circumvent this problem, we developed a strategy for reverse transcriptase real-time PCR that has considerable sensitivity and can be used to calculate relative changes in gene expression. This approach was validated in subregions of the rat cerebellum. Accordingly, expression of glial gene markers-myelin-associated glycoprotein and proteolipid protein 1-was found 70-160-fold higher in the white matter layer of the cerebellar cortex as compared to the neuron-enriched granular layer. In contrast, expression of a specific neuronal maker, neuron-specific enolase, was found seven-fold higher in the granular layer, as compared to the white matter layer. Furthermore, this approach had high sensitivity and specificity as we were able to detect a 38% decrease in the expression of neuron-specific enolase without a change in the expression of glial markers following administration of the neurotoxin, ibotenic acid. These results demonstrate feasibility of performing accurate semi-quantitative gene expression analyses in LCM samples. (c) 2005 Elsevier B.V. All rights reserved.