Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer

Dagmar M Kube,George G Klee,Farhad Kosari,George Vasmatzis,John C Cheville,Anne-Françoise Lamblin,Donald P Connelly,Cemile D Savci-Heijink

doi:10.1186/1471-2199-8-25

Abstract

BackgroundTo discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM) from prostate tissues and metastatic prostatic adenocarcinomas. Here we present methods developed, optimized, and validated to obtain high quality gene expression data.ResultsRNase inhibitor was included in solutions used to stain frozen tissue sections for LCM, which improved RNA quality significantly. Quantitative PCR assays, requiring minimal amounts of LCM RNA, were developed to determine RNA quality and concentration. SuperScript II™ reverse transcriptase was replaced with SuperScript III™, and SpeedVac concentration was eliminated to optimize linear amplification. The GeneChip® IVT labeling kit was used rather than the Enzo BioArray™ HighYield™ RNA transcript labeling kit since side-by-side comparisons indicated high-end signal saturation with the latter. We obtained 72 μg of labeled complementary RNA on average after linear amplification of about 2 ng of total RNA.ConclusionUnsupervised clustering placed 5/5 normal and 2/2 benign prostatic hyperplasia cases in one group, 5/7 Gleason pattern 3 cases in another group, and the remaining 2/7 pattern 3 cases in a third group with 8/8 Gleason pattern 5 cases and 3/3 metastatic prostatic adenocarcinomas. Differential expression of alpha-methylacyl coenzyme A racemase (AMACR) and hepsin was confirmed using quantitative PCR.

Highlights

To discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM) from prostate tissues and metastatic prostatic adenocarcinomas
Results of the analysis of RNA quality and concentration based on quantitative PCR (qPCR) with β-actin primers are presented in Fig. 2 for samples obtained after LCM of 27 prostate tissues
Since the 3'/M β-actin ratios were all two or lower, this was established as a quality control parameter for proceeding with linear amplification

Summary

Introduction

To discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM) from prostate tissues and metastatic prostatic adenocarcinomas. We present methods developed, optimized, and validated to obtain high quality gene expression data. Specific populations of benign and malignant cells were collected from frozen prostate tissues using laser capture microdis-. RNA was isolated from the cells and amplified to obtain sufficient quantities of labeled antisense RNA for microarray expression profiling. Methods upstream of RNA amplification, including frozen tissue processing, are crucial for preserving RNA integrity and obtaining accurate results from microarray experiments. A complete protocol for LCM, linear amplification of RNA, and microarray expression profiling is presented here. The protocol includes a quantitative PCR (qPCR) method for determining RNA concentration and integrity that is amenable to limited quantities of RNA obtained from LCM samples.

Methods

Results

Conclusion