Improvement of tissue preparation for laser capture microdissection: application for cell type-specific miRNA expression profiling in colorectal tumors.
Improvement of tissue preparation for laser capture microdissection: application for cell type-specific miRNA expression profiling in colorectal tumors.
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DOI：
10.1186/1471-2164-11-163
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发表时间：
2010-03-10
期刊：
影响因子：
4.4
通讯作者：
Zhu H
中科院分区：
文献类型：
--
作者：
Wang S;Wang L;Zhu T;Gao X;Li J;Wu Y;Zhu H
激光捕获显微切割（LCM）已成功地从组织切片中分离出纯细胞群，LCM与标准基因组学和蛋白质组学方法的结合彻底改变了复杂组织的分子分析。然而，LCM后回收的材料的数量和质量往往仍然是有限的分析使用全基因组和蛋白质组学方法。为了在LCM后获得高质量和数量的RNA，我们优化了组织制备的程序，并将该方法应用于结直肠肿瘤中的细胞类型特异性miRNA表达谱。我们发现，组织切片的乙醇固定2小时具有RNA质量（1.8倍，p = 0.0014）和数量（1.5倍，p = 0.066）的最大改善。总的来说，显微解剖结直肠组织的质量（RNA完整性数，RIN）对于正常（n = 43）、腺瘤（n = 14）和癌（n = 44）是5.2 ± 1.5（平均值± SD）、5.7 ± 1.1和7.2 ± 1.2。然后，我们使用Agilent miRNA微阵列比较了LCM选择的上皮细胞与基质细胞之间的18个结直肠组织（6个正常，6个腺瘤和6个癌）的miRNA表达谱。我们在这两种细胞类型之间鉴定了51个差异表达的miRNAs（p <= 0.001）。我们发现，上皮细胞中的miRNAs可以区分腺瘤与正常和癌。然而，间质细胞和混合细胞中的miRNAs不能将腺瘤与正常组织分开。最后，我们使用8个LCM选择的上皮细胞应用定量RT-PCR交叉验证7种不同miRNA的表达模式，发现两个平台之间的倍数变化具有良好的相关性（R = 0.996）。我们的研究证明了LCM与全基因组miRNA分析相结合在复杂组织中发现细胞类型特异性miRNA生物标志物的可行性和潜在能力。
Laser capture microdissection (LCM) has successfully isolated pure cell populations from tissue sections and the combination of LCM with standard genomic and proteomic methods has revolutionized molecular analysis of complex tissue. However, the quantity and quality of material recovered after LCM is often still limited for analysis by using whole genomic and proteomic approaches. To procure high quality and quantity of RNA after LCM, we optimized the procedures on tissue preparations and applied the approach for cell type-specific miRNA expression profiling in colorectal tumors. We found that the ethanol fixation of tissue sections for 2 hours had the maximum improvement of RNA quality (1.8 fold, p = 0.0014) and quantity (1.5 fold, p = 0.066). Overall, the quality (RNA integrity number, RIN) for the microdissected colorectal tissues was 5.2 ± 1.5 (average ± SD) for normal (n = 43), 5.7 ± 1.1 for adenomas (n = 14) and 7.2 ± 1.2 for carcinomas (n = 44). We then compared miRNA expression profiles of 18 colorectal tissues (6 normal, 6 adenomas and 6 carcinomas) between LCM selected epithelial cells versus stromal cells using Agilent miRNA microarrays. We identified 51 differentially expressed miRNAs (p <= 0.001) between these two cell types. We found that the miRNAs in the epithelial cells could differentiate adenomas from normal and carcinomas. However, the miRNAs in the stromal and mixed cells could not separate adenomas from normal tissues. Finally, we applied quantitative RT-PCR to cross-verify the expression patterns of 7 different miRNAs using 8 LCM-selected epithelial cells and found the excellent correlation of the fold changes between the two platforms (R = 0.996). Our study demonstrates the feasibility and potential power of discovering cell type-specific miRNA biomarkers in complex tissue using combination of LCM with genome-wide miRNA analysis.
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