miRNA Expression Analyses in Prostate Cancer Clinical Tissues

Summary

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

Abstract

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Introduction

Cancer of the prostate gland is a commonly diagnosed male malignancy that is one of the leading causes of cancer related mortality among men. In US, an estimated 220,800 new cases and 27,540 deaths will be reported in 2015¹.

Prostate cancer is a heterogeneous disease with highly variable disease course- tumors can be indolent or very aggressive. A critical challenge in prostate cancer clinical management is posed by the inadequacy of currently used methods/biomarkers for disease screening, diagnosis, prognosis and treatment². Current screening methods include prostate specific antigen (PSA) testing and a digital rectal....

Protocol

Formalin-fixed, paraffin-embedded (FFPE) or fresh frozen prostate cancer samples were obtained from the SFVAMC. Samples were from prostate cancer patients who underwent radical prostatectomy at SFVAMC. Written informed consent was obtained from all patients and the study was approved by the UCSF Committee on Human Research. Alternatively, prostate cancer tissues microarrays were procured from commercial sources and used for miRNA analyses by ISH. Clinicopathological and follow up information for analyzed prostate cancer patients was collected.

1. Tissue Samples

1. Cut prostate cancer tissue samples into 10 µm sections using a ....

Results

Expression profiling of miR-203 in LCM primary prostate cancer clinical specimens by RT-PCR analyses (Figure 1)

RT-PCR analyses of relative miR-203 expression in LCM primary prostate cancer tissues and the matched adjacent normal regions was carried out as described in Saini et al.¹⁵ RNU48 was used as a control. The Table below summarizes the relative miR-203 expression in prostate cancer tumor tissues relative to adjacent normal tissues.

Discussion

In this article, we describe a simplified workflow for miRNA expression profiling in archived FFPE or fresh frozen prostate cancer clinical tissues. In prostate cancer, several studies suggest an important role of microRNAs in prostate cancer initiation, progression and metastasis. However, conflicting results are often obtained on a specific miRNA²² since the miRNA extraction and analyses methods differ widely. In view of the emerging evidence supporting the potential application of miRNAs as alternative pros.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Microtome	Leica Biosystems	RM2255
Arcturus Autopix for LCM	Arcturus/ Life Technologies	LCM1621/LCM1110	Alternatively, Arcutus Xt system from Life Technolgies can be used.
CapSure Macro LCM Caps	Life Technologies	LCM0211
miRNeasy FFPE Kit	Qiagen	217504
7500 Fast Real-time PCR System	Applied Biosystems/ Life Technologies	4351106
Taqman MicroRNA Reverse Transcription kit	Applied Biosystems/ Life Technologies	4366596
Taqman Fast Universal PCR master mix	Applied Biosystems/ Life Technologies	4352042
DIG labeled LNA probe for U6	Exiqon	99002-01
BM Purple AP substrate	Roche	11442074001
Pre-hybridization solution	Biochain	K2191050-1
Hybridization solution	Biochain	K2191050-2
Blocking solution	Biochain	K2191050-8
AP-conjugated anti-digoxigenin antibody	Biochain	K2191050-7
Aqueous mounting media	Vector Laboratories	H-5501
Trizol (guanidine isothiocyanate-phenol reagent)	Life Technologies	15596-018
Harris hematoxylin	Statlab	SL200
Eosin	Statlab	SL201