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Quantitative expression profiling in formalin-fixed paraffin-embedded samples by affymetrix microarrays.

ABSTRACT: To date, few studies have systematically characterized microarray gene expression signal performance with degraded RNA from fixed (FFPE) in comparison with intact RNA from unfixed fresh-frozen (FF) specimens. RNA was extracted and isolated from paired tumor and normal samples from both FFPE and FF kidney, lung, and colon tissue specimens and microarray signal dynamics on both the raw probe and probeset level were evaluated. A contrast metric was developed to directly compare microarray signal derived from RNA extracted from matched FFPE and FF specimens. Gene-level summaries were then compared to determine the degree of overlap in expression profiles. RNA extracted from FFPE material was more degraded and fragmented than FF, resulting in a reduced dynamic range of expression signal. In addition, probe performance was not affected uniformly and declined sharply toward 5' end of genes. The most significant differences in FFPE versus FF signal were consistent across three tissue types and enriched with ribosomal genes. Our results show that archived FFPE samples can be used to profile for expression signatures and assess differential expression similar to unfixed tissue sources. This study provides guidelines for application of these methods in the discovery, validation, and clinical application of microarray expression profiling with FFPE material.

SUBMITTER: Abdueva D

PROVIDER: S-EPMC2893624 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Quantitative expression profiling in formalin-fixed paraffin-embedded samples by affymetrix microarrays.

Abdueva Diana D Wing Michele M Schaub Betty B Triche Timothy T Davicioni Elai E

The Journal of molecular diagnostics : JMD 20100603 4

To date, few studies have systematically characterized microarray gene expression signal performance with degraded RNA from fixed (FFPE) in comparison with intact RNA from unfixed fresh-frozen (FF) specimens. RNA was extracted and isolated from paired tumor and normal samples from both FFPE and FF kidney, lung, and colon tissue specimens and microarray signal dynamics on both the raw probe and probeset level were evaluated. A contrast metric was developed to directly compare microarray signal de ...[more]

PMID: 20522636

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Project description:BackgroundWe have developed a gene expression assay (Whole-Genome DASL), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens.Methodology/principal findingsWe demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3-1.5 and 1.5-2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2) approximately 0.86 and R(2) approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R(2) approximately 0.92), with approximately 71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R(2) approximately 0.80, while still maintaining a correlation of R(2) approximately 0.75 with standard FFPE inputs (200 ng).Conclusions/significanceTaken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens).

Project description:Endometriosis is an inflammatory pathology associated with a negative effect on life quality. Recently, this pathology was connected to ovarian cancer, in particular with endometrioid ovarian cancer. microRNAs (miRNAs) are a class of RNA transcripts ~19-22 nucleotides in length, the altered miRNA pattern being connected to pathological status. miRNAs are highly stable transcripts, and these can be assessed from formalin-fixed paraffin-embedded (FFPE) samples leading to the identification of miRNAs that could be developed as diagnostic and prognostic biomarkers, in particular those involved in malignant transformation. The aim of our study was to evaluate miRNA expression pattern in FFPE samples from endometriosis and ovarian cancer patients using PCR-array technology and also to compare the differential expression pattern in ovarian cancer versus endometriosis. For the PCR-array study, we have used nine macrodissected FFPE samples from endometriosis tissue, eight samples of ovarian cancers and five normal ovarian tissues. Quantitative real-time PCR (qRT-PCR) was used for data validation in a new patient cohort of 17 normal samples, 33 endometriosis samples and 28 ovarian cancer macrodissected FFPE samples. Considering 1.5-fold expression difference as a cut-off level and a P-value <0.05, we have identified four miRNAs being overexpressed in endometrial tissue, while in ovarian cancer 15 were differentially expressed (nine overexpressed and six downregulated). The expression level was confirmed by qRT-PCR for miR-93, miR-141, miR-155, miR-429, miR-200c, miR-205 and miR-492. Using the interpretative program Ingenuity Pathway Analysis revealed several deregulated pathways due to abnormal miRNA expression in endometriosis and ovarian cancer, which in turn is responsible for pathogenesis; this differential expression of miRNAs can be exploited as a therapeutic target. A higher number of altered miRNAs were detected in endometriosis versus ovarian cancer tissue, most of them being linked with epithelial-to-mesenchymal transition.

Dataset Information

Quantitative expression profiling in formalin-fixed paraffin-embedded samples by affymetrix microarrays.

Publications

Quantitative expression profiling in formalin-fixed paraffin-embedded samples by affymetrix microarrays.

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