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

journal contribution
posted on 2023-05-19, 15:10 authored by Liesel FitzgeraldLiesel Fitzgerald, Jung, C-H, Wong, EM, Joo, JE, Gould, JA, Vasic, V, Bassett, JK, O'Callaghan, N, Nottle, T, Pedersen, J, Giles, GG, Southey, MC
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 µm); 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.

History

Publication title

Laboratory Investigation

Volume

98

Issue

4

Pagination

537-550

ISSN

0023-6837

Department/School

Menzies Institute for Medical Research

Publisher

Lippincott Williams & Wilkins

Place of publication

530 Walnut St, Philadelphia, USA, Pa, 19106-3621

Rights statement

Copyright 2018 United States & Canadian Academy of Pathology

Repository Status

  • Restricted

Socio-economic Objectives

Clinical health not elsewhere classified

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