Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

A novel method of amplification of FFPET derived-RNA enables accurate disease classification with microarrays

ABSTRACT: A new method for amplification and labeling of RNA is assessed that permits gene expression microarray analysis of formalin-fixed paraffin embedded tissue (i.e. FFPET) samples. Valid biological data was obtained using gene expression microarrays of diffuse large B-cell lymphoma (i.e. DLBCL) FFPET samples. We examined 59 matched DLBCL patient samples, FFPET and fresh/frozen. The samples contained both prognostic subgroups of DLBCL: germinal center B-cell (i.e. GCB) and activated B-cell (ABC). Fresh/frozen (i.e. FF) samples were amplified by both the traditional Eberwine oligo-dT method and a new method described herein. The matching FFPET samples were also amplified using the new method. This study is a collaboration among Roche Molecular Diagnostics, British Columbia Cancer Agency, National Cancer Institute and Nugen Technologies (the maker of the WT-Ovation FFPET reagents).

ORGANISM(S): Homo sapiens

DISEASE(S): B-cell lymphoma

SUBMITTER: Rachel Li

PROVIDER: E-GEOD-19246 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A novel method of amplification of FFPET-derived RNA enables accurate disease classification with microarrays.

Williams P Mickey PM Li Rui R Johnson Nathalie A NA Wright George G Heath Joe-Don JD Gascoyne Randy D RD

The Journal of molecular diagnostics : JMD 20100805 5

A new method for amplification and labeling of RNA is assessed that permits gene expression microarray analysis of formalin-fixed paraffin-embedded tissue (FFPET) samples. Valid biological data were obtained using gene expression microarrays of diffuse large B-cell lymphoma (DLBCL) FFPET samples. We examined 59 matched DLBCL patient samples, FFPET, and fresh/frozen. The samples contained both prognostic subgroups of DLBCL: germinal center B-cell (GCB) and activated B-cell (ABC). Fresh/frozen (FF ...[more]

PMID: 20688907

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Project description:This study was performed to evaluated RNA extraction and gene expression analysis of FFPE specimen stored for more than 20 years. Using long time stored FFPE material; large retrospective studies correlating molecular features with therapeutic response and clinical outcome, can be performed. Quantitative PCR (qPCR) was used to evaluate RNA extraction methods and to compare gene expression profiles of FFPE and fresh frozen (FF) tissue. Extracted RNA was subsequently subjected to microarray analysis and compared to qPCR data. The Ambion RecoverAll kit appears to be particularly suited for RNA extraction of long time stored FFPE tissues. Gene expression analysis using Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues. We conclude that high quality gene expression signatures can be recognized using Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality. Three RNA extraction methods from Roche (Basel, Switzerland), Ambion (Austin, TX, USA) and Qiagen (Hilden, Germany), designed for FFPE material was used. The methods were compared using qPCR. For the qPCR analysis, two different concentrations of input cDNA were used (20ng and 100ng) and 32 human endogenous control genes were examined. RNA from the Ambion FFPE kit was further analyzed by using microarray. Amplification of RNA prior to microarray analysis was performed using Nugen technologies (San Carlos, CA, USA). Nugen has developed amplification kits both for FFPE and FF materials: WT- ovation FFPE RNA amplification System V2 (Nugen FFPE), Ovation FF RNA Amplification System V2 (Nugen V2 FF) and Ovation FF Pico RNA Amplification System (Nugen PICO FF). The Nugen FFPE kit, designed for FFPE material was only used for FFPE tissue. The Nugen Pico FF kit, designed to target small amounts of FF RNA (>500 pg) and the Nugen V2 FF kit designed to target total FF RNA, were only used for FF tissue. Affymetrix standard amplification protocol (Affy FF) designed for FF RNA was also included as the standard method for amplification.

Dataset Information

A novel method of amplification of FFPET derived-RNA enables accurate disease classification with microarrays

Publications

A novel method of amplification of FFPET-derived RNA enables accurate disease classification with microarrays.

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