人类前列腺上皮的激光捕获显微切割的RNA分析
Summary
The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.
Abstract
The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.
Introduction
前列腺是一种异质性组织安排在腺体腺泡由肌纤维间质主要由平滑肌组成1包围分泌上皮细胞组成。上皮隔室由五个不同但有组织的细胞类型：基底细胞，分泌细胞，神经内分泌细胞，短暂扩充细胞和干细胞2。在前列腺癌（PCa），它产生于腔上皮细胞，腺癌的生长会导致基质室 3的一个明显的渐进性下降。由于这些原因，组织标本将具有在基质和上皮细胞类型的基于前列腺癌的程度的比例有明显的差异。这些差异会导致从整个组织，不考虑所期望的细胞类型的显微解剖获得的基因表达数据的偏颇的假设。因此，为了消除这种偏差，必须事先对RNA提取和分离的细胞类型基因表达分析。
Macrodissection或显微切割可用于物理地从周围基质4 -6分离充分表征的上皮区域。 Macrodissection典型的做法是与在解剖显微镜下用刀片和可以很好地用于分离大前列腺癌从基质结节，但是不能够从周围的基质中除去良性上皮的（参见实施例良性前列腺组织学于图1）。显微切割用激光（LCM）比macrodissection显著劳动密集型的，但可以非常精确地解剖良性上皮4。
从我们的实验室最近的出版物已经表明，核糖核酸可以成功地通过从任一福尔马林固定石蜡包埋（FFPE）的活检或冷冻的组织4,7-9 LCM萃取。在LCM-RNA提取的主要挑战是：1）精确解剖组织的所需区域，和2）以保持ř液晶显示模块和分离过程4,10中NA的完整性。从纯的细胞群中分离RNA可通过多种方法，包括反转录定量PCR（RT-qPCR的）7,8，微阵列11，和深-sequencing 12-14中可用于基因表达分析。
该协议的目的是从隔离LCM前列腺上皮的总RNA从冷冻的组织为下游基因表达分析。
Protocol
Representative Results
Discussion
基因表达的来自人的标本分析可以是具有挑战性的，不仅对质量或组织的可用量，同时也为存在于一个给定的组织标本的各种组织学实体。这是在其中良性组织基本上是间质组织和癌症领域是不含基质的前列腺特别具有挑战性。 LCM促进前列腺间质和上皮的RNA物理分离为两种不同的细胞类型（图1A）的更精确的签名。在比较macrodissection或全部组织处理，LCM可以分离细胞区室中良性组织?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Vicky Macias, Angeline Giangreco and Avani Vaishnav for assistance with optimizing this methodology over the years and Yang Zhang and Dr. Jian Ma at the University of Illinois at Urbana-Champaign for the RNA seq analysis. This work was supported by NIH/NCI R01 CA166588-01 (Nonn), American Cancer Society Research Scholar 124264-RSG-13-012-01-CNE (Nonn), NIH/NCI R03 CA172827-01 (Nonn), DOD-CDMPR PRCP Health Disparities Idea Award PC121923 (Nonn) and a Prevent Cancer Foundation grant (Zhou).
Materials
| RNase-AWAY | MBP | 7005-11 | |
| PEN-membrane 4,0 mm slides | Leica | 11600289 | |
| Glass slides, Superfrost Plus | FisherBrand | 12-550-15 | |
| Ethanol 200 proof | Decon labs. | 2701 | |
| DEPC (diethyl pyrocarbonate) | Sigma | D-5758 | |
| Cryostat | Leica | CM3050 | |
| Coplins (Staining jar) | IHCWORLD | M900-12 | |
| Coplins (Staining rack) | IHCWORLD | M905-12 | |
| Aperio ScanScope | Aperio(Leica) | ScanScope® CS | |
| Toluidine Blue | Fluka | 89640-5G | |
| Laser Microdissection System | Leica | LMD7000 | |
| 0.5 mL Thin-walled Tubes for LCM | Thermo Scientific | AB-0350 | |
| RNAqueous®-Micro Total RNA Isolation Kit | Ambion | AM1931 | Thermo Fisher Scientific Brand |
| NanoDrop | Thermo Scientific | ND-1000 | |
| Qubit 2.0 Fluorometer | Life Technologies | Q32866 | Thermo Fisher Scientific Brand |
| High-Capacity cDNA Reverse Transcription Kit | Applied Biosystems | 4368814 | Thermo Fisher Scientific Brand |
| Universal cDNA Synthesis Kit II, 8-64 rxns | Exiqon | 203301 | |
| TaqMan microRNA RT kit | Applied Biosystems | 4366597 | Thermo Fisher Scientific Brand |
| Hematoxylin stain | Ricca Chemical Company | 3536-16 | |
| Eosin-Y | Richard Allan Scientific | 7111 |
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