Laser-fangst mikrodisseksjon of Human prostata Epitel for RNA-analyse
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
Prostatakjertelen er en heterogen vev består av sekretorisk epitel arrangert i kjertel acini omgitt av fibromuscular stroma består hovedsakelig av glatt muskulatur en. Epithelial rommet består av fem forskjellige men organiserte celletyper: basal celler, sekretoriske celler, nevroendokrine celler, transitt forsterke celler og stamceller 2. I prostata cancer (PCA), som oppstår fra den luminale epitelceller, fører til vekst av adenokarsinom en tydelig progressive reduksjonen av stromal kammeret 3. Av disse grunner, vil vevsprøver har distinkte forskjeller i andelen av stromal og epitelial celletype basert på omfanget av PCa. Disse forskjellene kan føre til forutinntatte antagelser om genuttrykk data innhentet fra hele vev uten hensyn til mikrodisseksjon av den ønskede celletype. Derfor, for å fjerne denne skjevheten er det viktig å skille celletyper før RNA ekstraksjon oggenekspresjonsanalyser.
Macrodissection eller microdissection kan brukes til å fysisk separere velkarakteriserte epitel områder fra den omkringliggende stroma 4 -6. Macrodissection gjøres typisk med et barberblad under et disseksjonsmikroskop og fungerer godt for separasjon av store PCa knuter fra stroma, men er ikke i stand til å fjerne benign epitel fra omkringliggende stroma (se eksempel benign prostata histologi i figur 1). Mikrodisseksjon med en laser (LCM) er vesentlig mer arbeidskrevende enn macrodissection, men kan svært nøyaktig dissekere godartet epitel fire.
Nye publikasjoner fra vårt laboratorium har vist at RNA kan med hell hentet av LCM fra enten formalinfiksert parafin-embedded (FFPE) biopsier eller frosset vev 4,7-9. De store utfordringene i LCM-RNA ekstraksjon er 1) å nøyaktig dissekere de ønskede områder av vev, og 2) å bevare RNA integritet under LCM og isolasjonsprosess 4,10. RNA isolert fra rene cellepopulasjoner kan brukes genuttrykk analyse for ved flere metoder inkludert revers-transkripsjon kvantitativ PCR (RT-qPCR) 7,8, mikromatriser 11, og dype -sequencing 12-14.
Målet med denne protokollen er å isolere total RNA fra LCM prostatahyperplasi epitel fra frosset vev for nedstrøms genekspresjon analyser.
Protocol
Representative Results
Discussion
Genekspresjon profilering fra humane prøvene kan være utfordrende, ikke bare for den kvalitet eller kvantitet av vev er tilgjengelig, men også for de ulike histologiske-enhetene i en gitt vevsprøve. Dette er spesielt utfordrende i prostata hvor godartet vev er i stor grad stromale vev og områder av kreft er blottet for stroma. LCM muliggjør fysisk separasjon av prostatisk stroma og epitel RNA for en mer nøyaktig signatur av de to forskjellige celletyper (figur 1A). I forhold til macrodissection e…
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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