cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue
cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue
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DOI：
10.1016/s0002-9440(10)64352-0
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发表时间：
2002-01-01
影响因子：
6
通讯作者：
Bohle, RM
中科院分区：
文献类型：
--
作者：
Fink, L;Kohlhoff, S;Bohle, RM
利用cDNA芯片可以有效地研究基因的差异表达。由于组织匀浆不可避免地导致大量不同细胞的平均表达，我们的目标是将联合收割机mRNA分析与细胞类型特异性显微切割相结合。使用聚合酶链反应（PCR）为基础的预扩增技术，表达谱被证明是保存。我们修改了现有的协议，使其能够应用从显微切割的细胞中提取的RNA的总量。接近1000的平均扩增因子允许将初始RNA的需求减少到接近10 ng。该技术用于研究小鼠肺内动脉（类似于500个细胞当量）。使用具有1176个斑点的过滤器，三个独立的实验显示了预扩增的cDNA的表达的高度一致性。这些特征主要不同于总肺匀浆的特征。此外，在实验性低氧诱导的肺动脉高压，扩增的cDNA从这些肺内血管的cDNA进行了比较，从血管解剖从含氧量正常的肺。通过将显微切割与实时聚合酶链反应（PCR）连接，获得了替代方法的验证。如阵列数据所示，通过PCR技术完全证实了具有不同上调因子的9个选定基因。因此，提出了一种快速的协议相结合的显微切割和阵列分析，需要低数量的初始RNA，以评估可靠的细胞类型特异性基因调控，即使在非肿瘤性复杂组织。
Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to similar to10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs (similar to500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from normoxic lungs. Validation by an alternative method was obtained by linking microdissection with real-time polymerase chain reaction (PCR). As suggested by the array data, nine selected genes with different factors of up-regulation were fully confirmed by the PCR technique. Thus, a rapid protocol is presented combining microdissection and array profiling that demands low quantities of initial RNA to assess reliably cell-type-specific gene regulation even within nonneoplastic complex tissues.