Abstract 1836: Global gene expression profiles from bladder tumor FFPE samples
Authors: Bagai Varun , Schageman Jeoffrey , Bramlett Kelli , McConkey David J. , Choi Woonyoung 
Published: July 2016

Cancer is a disease characterized by uncontrolled cell growth and proliferation. Recent advances in molecular medicine and cancer biology have changed the way clinicians evaluate and consider treatment. Selected tumor biomarkers have been utilized as targets for drug therapy leading to better more effective treatment. Gene expression profiling has been used for identifying new biomarkers for tumor classification and driving decision making for better patient outcome in different tumor types. DNA microarrays have become a key method to acquire a comparative snapshot of the gene expression profile from test samples in a high throughput manner. Quantitative PCR and newer sequencing techniques are popular alternatives offering highly accurate gene expression measurements, but with limitations due to cost, complex instrumentation and analysis needs. RNA extracted from formalin fixed paraffin embedded tissue (FFPE) creates considerable additional challenges in acquiring accurate gene expression measurements due to the highly fragmented and compromised integrity of FFPE RNA due to the fixation process.

To address the challenges of current sequencing based methods and take advantage of the simplicity of analysis that comes with using technologies such as microarrays; we have tested the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit using RNA isolated from bladder tumor FFPE specimens. This targeted RNA sequencing approach allows profiling the global mRNA expression of human RNA in a highly multiplexed fashion using the Ion AmpliSeq™ technology. 10ng of total RNA extracted from FFPE tissue was reverse transcribed followed by automated library preparation on the Ion Chef™ system using the new Ion AmpliSeq™ Kit for Chef and the Ion AmpliSeq™ Transcriptome Human Gene Expression Panel. Eight pooled libraries were then sequenced on the Ion S5™XL System with Ion 540™ Chip. Libraries were also prepared with well characterized control RNAs, Universal Human Reference RNA (UHR) and First Choice Human Brain Reference RNA (HBR) using both the manual and automated library generation protocol for validation and comparison studies.

The results show detection of more genes than popular microarray platforms with comparable differential gene expression measurements to quantitative PCR (r = 0.96) and RNA-Seq methods (r = 0.94). Gene expression values correlated with R&gt;0.99 for all technical replicates and R&gt;0.95 between manual and automated library preparation methods using well characterized samples. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit is a simple method to measure global gene expression profiles from human RNA samples in a timely, cost effective, and high throughput manner resulting in sensitive and accurate gene expression measurements. The new S5™XL System combined with automated library and template preparation on the Ion Chef™ system enable a simple RNA to gene expression data workflow requiring only 45 minutes of hands on time from 10ng of FFPE RNA.

Citation Format: Varun Bagai, Jeoffrey Schageman, Kelli Bramlett, David J. McConkey, Woonyoung Choi. Global gene expression profiles from bladder tumor FFPE samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1836.