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Obtaining high quality transcriptome data from formalin-fixed, paraffin-embedded diagnostic prostate tumor specimens

Citation

FitzGerald, LM and Jung, C-H and Wong, EM and Joo, JE and Gould, JA and Vasic, V and Bassett, JK and O'Callaghan, N and Nottle, T and Pedersen, J and Giles, GG and Southey, MC, Obtaining high quality transcriptome data from formalin-fixed, paraffin-embedded diagnostic prostate tumor specimens, Laboratory Investigation pp. 1-14. ISSN 0023-6837 (2018) [Refereed Article]

Copyright Statement

Copyright 2018 United States & Canadian Academy of Pathology

DOI: doi:10.1038/s41374-017-0001-8

Abstract

Prognostic genomic biomarkers that can be measured at diagnosis to aid choice of treatment options are unavailable for most common cancers. This is due in part to the poor quality and quantity of available diagnostic specimens for discovery research and to limitations in genomic technologies. Recent technical advances now enable high-density molecular analyses using suboptimal biological specimens. Here we describe the optimization of a transcriptome-specific protocol for use with formalin-fixed, paraffin-embedded (FFPE) diagnostic prostate cancer (PrCa) specimens. We applied the Ion AmpliSeq Transcriptome Human Gene Expression Kit (AmpliSeq Kit) to RNA samples extracted from 36 tumor-enriched and 16 adjacent normal tissues (ADJNT) from 37 FFPE PrCa specimens over a series of eight pilot studies, incorporating protocol modifications from Pilots 2 to 5. Data quality were measured by (1) the total number of mapped reads; (2) the percentage of reads that mapped to AmpliSeq target regions (OnTarget%); (3) the percentage of genes on the AmpliSeq panel with a read count ≥10 (TargetsDetected%); and (4) comparing the gene read-count distribution of the prostate tissue samples with the median gene read-count distribution of cell line-derived RNA samples. Modifications incorporated into Pilot study 5 provided gene expression data equivalent to cell line-derived RNA samples. These modifications included the use of freshly cut slides for macrodissection; increased tissue section thickness (8 Ám); RNA extraction using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (ThermoFisher); 18 target amplification cycles; and processing six samples per Ion PI chip. This protocol will facilitate the discovery of prognostic biomarkers for cancer by allowing researchers to exploit previously underutilized diagnostic FFPE specimens.

Item Details

Item Type:Refereed Article
Keywords:prostate cancer, gene expression, methodology, FFPE
Research Division:Medical and Health Sciences
Research Group:Oncology and Carcinogenesis
Research Field:Cancer Genetics
Objective Division:Health
Objective Group:Clinical Health (Organs, Diseases and Abnormal Conditions)
Objective Field:Cancer and Related Disorders
Author:FitzGerald, LM (Dr Liesel Fitzgerald)
ID Code:123728
Year Published:2018
Deposited By:Menzies Institute for Medical Research
Deposited On:2018-01-22
Last Modified:2018-05-07
Downloads:0

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