Dataset Information

Expression profiling of archival tumors for long-term health studies.

ABSTRACT: PURPOSE:More than 20 million archival tissue samples are stored annually in the United States as formalin-fixed, paraffin-embedded (FFPE) blocks, but RNA degradation during fixation and storage has prevented their use for transcriptional profiling. New and highly sensitive assays for whole-transcriptome microarray analysis of FFPE tissues are now available, but resulting data include noise and variability for which previous expression array methods are inadequate. EXPERIMENTAL DESIGN:We present the two largest whole-genome expression studies from FFPE tissues to date, comprising 1,003 colorectal cancer (CRC) and 168 breast cancer samples, combined with a meta-analysis of 14 new and published FFPE microarray datasets. We develop and validate quality control (QC) methods through technical replication, independent samples, comparison to results from fresh-frozen tissue, and recovery of expected associations between gene expression and protein abundance. RESULTS:Archival tissues from large, multicenter studies showed a much wider range of transcriptional data quality relative to smaller or frozen tissue studies and required stringent QC for subsequent analysis. We developed novel methods for such QC of archival tissue expression profiles based on sample dynamic range and per-study median profile. This enabled validated identification of gene signatures of microsatellite instability and additional features of CRC, and improved recovery of associations between gene expression and protein abundance of MLH1, FASN, CDX2, MGMT, and SIRT1 in CRC tumors. CONCLUSIONS:These methods for large-scale QC of FFPE expression profiles enable study of the cancer transcriptome in relation to extensive clinicopathological information, tumor molecular biomarkers, and long-term lifestyle and outcome data.

SUBMITTER: Waldron L

PROVIDER: S-EPMC3500412 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Expression profiling of archival tumors for long-term health studies.

Waldron Levi L Ogino Shuji S Hoshida Yujin Y Shima Kaori K McCart Reed Amy E AE Simpson Peter T PT Baba Yoshifumi Y Nosho Katsuhiko K Segata Nicola N Vargas Ana Cristina AC Cummings Margaret C MC Lakhani Sunil R SR Kirkner Gregory J GJ Giovannucci Edward E Quackenbush John J Golub Todd R TR Fuchs Charles S CS Parmigiani Giovanni G Huttenhower Curtis C

Clinical cancer research : an official journal of the American Association for Cancer Research 20121107 22

<h4>Purpose</h4>More than 20 million archival tissue samples are stored annually in the United States as formalin-fixed, paraffin-embedded (FFPE) blocks, but RNA degradation during fixation and storage has prevented their use for transcriptional profiling. New and highly sensitive assays for whole-transcriptome microarray analysis of FFPE tissues are now available, but resulting data include noise and variability for which previous expression array methods are inadequate.<h4>Experimental design< ...[more]

PMID: 23136189

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Project description:Advantages of RNA-Seq over array based platforms are quantitative gene expression and discovery of expressed single nucleotide variants (eSNVs) and fusion transcripts from a single platform, but the sensitivity for each of these characteristics is unknown. We measured gene expression in a set of manually degraded RNAs, nine pairs of matched fresh-frozen, and FFPE RNA isolated from breast tumor with the hybridization based, NanoString nCounter (226 gene panel) and with whole transcriptome RNA-Seq using RiboZeroGold ScriptSeq V2 library preparation kits. We performed correlation analyses of gene expression between samples and across platforms. We then specifically assessed whole transcriptome expression of lincRNA and discovery of eSNVs and fusion transcripts in the FFPE RNA-Seq data. For gene expression in the manually degraded samples, we observed Pearson correlations of >0.94 and >0.80 with NanoString and ScriptSeq protocols, respectively. Gene expression data for matched fresh-frozen and FFPE samples yielded mean Pearson correlations of 0.874 and 0.783 for NanoString (226 genes) and ScriptSeq whole transcriptome protocols respectively, p<2x10(-16). Specifically for lincRNAs, we observed superb Pearson correlation (0.988) between matched fresh-frozen and FFPE pairs. FFPE samples across NanoString and RNA-Seq platforms gave a mean Pearson correlation of 0.838. In FFPE libraries, we detected 53.4% of high confidence SNVs and 24% of high confidence fusion transcripts. Sensitivity of fusion transcript detection was not overcome by an increase in depth of sequencing up to 3-fold (increase from ~56 to ~159 million reads). Both NanoString and ScriptSeq RNA-Seq technologies yield reliable gene expression data for degraded and FFPE material. The high degree of correlation between NanoString and RNA-Seq platforms suggests discovery based whole transcriptome studies from FFPE material will produce reliable expression data. The RiboZeroGold ScriptSeq protocol performed particularly well for lincRNA expression from FFPE libraries, but detection of eSNV and fusion transcripts was less sensitive.

Project description:BACKGROUND: It is believed that the main factors of low prenatal growth in mammals are genetic and environmental. We used isogenic mice maintained in standard conditions to analyze how natural non-genetic microsomia (low birth weight) is produced in inbred mice and its long term effect on health. To better understand the molecular basis of non-genetic microsomia, we undertook transcriptome profiling of both male and female livers from small and normal size mice at birth. RESULTS: Naturally occurring neonatal microsomia was defined as a gender-specific weanling weight under the 10th percentile of the colony. Birth weight variation was similar in inbred and outbred lines. Mice were phenotyped by weight, size, blood pressure, organ size, their response to a glucose challenge, and survival rates. Regardless of diet, adult mice born with microsomia showed a significantly lower body weight and size, and differences in the weight of several organs of microsomic adult mice compared to normal birth weight adults were found. After a high-fat diet, microsomic mice were less prone to obesity, showing a better glucose tolerance and lower blood pressure. Through a transcriptome analysis, we detected a different pattern of mRNA transcription in the liver at birth comparing male vs female and microsomic vs normal mice, noting some modifications in epigenetic regulatory genes in females and modifications in some growth factor genes in males. Finally, using embryo transfer of embryos of different quality and age, we identified a putative preimplantation origin of this non-genetic microsomia. CONCLUSIONS: (1) neonatal microsomia is not always a risk factor for adult metabolic syndrome, (2) neonatal non-genetic microsomia displays changes in the expression of important epigenetic genes and changes in liver mRNA transcription profile at birth, exaggerating sexual dimorphism, and (3) random preimplantation phenotypic variability could partially explain body birth weight variation in isogenic lines.

Dataset Information

Expression profiling of archival tumors for long-term health studies.

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Expression profiling of archival tumors for long-term health studies.

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