RNA分析のためのヒト前立腺上皮のレーザーキャプチャーマイクロダイセクション
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
前立腺は、主に平滑筋1で構成される線維筋間質に囲まれた腺房に配置された分泌上皮で構成される異種の組織です。基底細胞、分泌細胞、神経内分泌細胞、トランジット増幅細胞と幹細胞の2：上皮の区画は、5つの異なるが、組織的な細胞型で構成されています。管腔の上皮細胞から生じる前立腺癌（PCA）では、腺癌の成長は、間質コンパートメント3の明白な進行性の低下の原因となります。これらの理由のために、組織標本は、PCaの程度に基づいて、間質および上皮細胞型の割合の明確な違いがあります。これらの差異は、所望の細胞型の顕微解剖に関係なく、全体の組織から取得した遺伝子発現データのバイアスされた仮定を導くことができます。したがって、このバイアスを除去するために、RNA抽出前に別個の細胞型に必須であると遺伝子発現解析。
Macrodissectionまたは顕微解剖は、物理的に周囲の間質4 -6から十分に特徴付け上皮領域を分離するために使用することができます。 Macrodissectionは通常、解剖顕微鏡下でのかみそりの刃で行われ、大規模なPCAが間質から結節分離に適していますが、間質を取り巻くから良性の上皮を除去することができるではありません（ 図1の良性前立腺組織像の例を参照）。レーザー（LCM）と顕微解剖はmacrodissectionよりもはるかに多くの労働集約的であるが、非常に正確に良性の上皮4を分析することができます。
我々の研究室からの最近の刊行物は、RNAが正常にホルマリン固定パラフィン包埋（FFPE）生検または凍結組織4,7-9のいずれかからLCMによって抽出することができることを示しています。 LCM-RNA抽出の主要な課題は1である）を正確に組織の所望の領域を分析するため、及び2）Rを保存しますLCMおよび分離プロセス4,10の間に整合性NA。純粋な細胞集団から単離したRNAを逆転写定量的PCR（RT-qPCRの）7,8、マイクロアレイ11、及び深い-sequencing 12-14を含むいくつかの方法によって、遺伝子発現解析のために使用することができます。
このプロトコルの目的は、下流の遺伝子発現分析のために凍結した組織からのLCMの前立腺上皮からの全RNAを単離することです。
Protocol
Representative Results
Discussion
人間の標本から遺伝子発現プロファイリングは、利用可能な組織の品質や数量のでなく、与えられた組織標本に存在する様々な組織学的実体のためだけではなく、挑戦することができます。これは、良性組織が大きく、間質組織および癌の領域である間質を欠いていた前立腺では特に困難です。 LCMは、2つの異なる細胞タイプ（ 図1A）のより正確な署名のための前立腺間質および…
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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