Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

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

The prostate gland is a heterogeneous tissue composed of secretory epithelium arranged in glandular acini surrounded by fibromuscular stroma composed primarily of smooth muscle¹. The epithelial compartment is comprised of five different but organized cell types: basal cells, secretory cells, neuroendocrine cells, transit amplifying cells and stem cells². In prostate cancer (PCa), which arises from the luminal epithelial cells, the growth of the adenocarcinoma causes an evident progressive decline of the stromal compartment³. For these reasons, tissue specimens will have distinct differences in the proportion of stromal and epithelial c....

Protocol

All human tissues used for these experiments were acquired via an Institutional Review Board approved protocol and/or exemption at University of Illinois at Chicago.

1. Section Fresh Frozen Prostate onto PEN-slide and onto Charged Glass Slide

1. The day before or a few hours before sectioning the sample, clean tools (i.e., brush, forceps, coplins, blades, PEN-framed slides (if not already RNase free), an ETOH safe marker and a pencil) and the inside of a RT cryostat with RNase-decontaminating solution using a spray bottle and lab-wipes.
   1. Allow RNase-decontaminating solution to sit for 5 min before wiping away, rinse with depcH_2....

Results

In a previous study we demonstrated the use of LCM to collect epithelial and stromal tissues to compare expression profiling by RT-qPCR of mRNA and microRNAs from frozen and FFPE prostate tissues from the same patient⁴. LCM is time consuming, particularly if large amount of RNA is to be collected for next-generation sequencing analysis. Therefore, it is crucial to keep the working space and tools RNase free. It is recommended to examine quality and cell-specificity controls in the RNA collected (discussed in m.......

Discussion

Gene expression profiling from human specimens can be challenging, not only for the quality or quantity of tissue available, but also for the various histological entities present in a given tissue specimen. This is particularly challenging in the prostate in which benign tissues are largely stromal tissues and areas of cancer are devoid of stroma. LCM facilitates physical separation of prostatic stroma and epithelium RNA for a more accurate signature of the two different cell types (Figure 1A). In compa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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