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

Autor: FitzGerald LM; Cancer Epidemiology and Intelligence Division, Cancer Council Victoria, Melbourne, VIC, Australia.; Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia., Jung CH; Melbourne Bioinformatics, The University of Melbourne, Parkville, VIC, Australia., Wong EM; Department of Pathology, The University of Melbourne, Parkville, VIC, Australia.; Precision Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Australia., Joo JE; Department of Pathology, The University of Melbourne, Parkville, VIC, Australia.; Precision Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Australia., Gould JA; Monash Health Translation Precinct, Medical Genomics Facility, Hudson Institute of Medical Research, Clayton, VIC, Australia., Vasic V; Monash Health Translation Precinct, Medical Genomics Facility, Hudson Institute of Medical Research, Clayton, VIC, Australia., Bassett JK; Cancer Epidemiology and Intelligence Division, Cancer Council Victoria, Melbourne, VIC, Australia., O'Callaghan N; Department of Pathology, The University of Melbourne, Parkville, VIC, Australia., Nottle T; TissuPath Specialist Pathology, Mount Waverley, VIC, Australia., Pedersen J; TissuPath Specialist Pathology, Mount Waverley, VIC, Australia., Giles GG; Cancer Epidemiology and Intelligence Division, Cancer Council Victoria, Melbourne, VIC, Australia.; Centre for Epidemiology and Biostatistics, School of Global and Population Health, The University of Melbourne, Melbourne, VIC, Australia., Southey MC; Cancer Epidemiology and Intelligence Division, Cancer Council Victoria, Melbourne, VIC, Australia. msouthey@unimelb.edu.au.; Department of Pathology, The University of Melbourne, Parkville, VIC, Australia. msouthey@unimelb.edu.au.; Precision Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Australia. msouthey@unimelb.edu.au.
Jazyk: angličtina
Zdroj: Laboratory investigation; a journal of technical methods and pathology [Lab Invest] 2018 Apr; Vol. 98 (4), pp. 537-550. Date of Electronic Publication: 2018 Jan 16.
DOI: 10.1038/s41374-017-0001-8
Abstrakt: 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 (ADJ NT ) 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.
Databáze: MEDLINE
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