Dataset Information

FFPE - Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics

ABSTRACT: protein extraction from FFPE tissue comparison

INSTRUMENT(S): LTQ Orbitrap, instrument model, 4700 Proteomics Analyzer

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hakan Sarioglu

PROVIDER: PRD000234 | Pride | 2011-05-05

REPOSITORIES: Pride

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Publications

Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics.

Azimzadeh Omid O Barjaktarovic Zarko Z Aubele Michaela M Calzada-Wack Julia J Sarioglu Hakan H Atkinson Michael J MJ Tapio Soile S

Journal of proteome research 20100901 9

Formalin-fixed paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the fixation process, proteins undergo degradation and cross-linking, making conventional protein analysis technologies problematic. In this study, we have compared several extraction and separation methods for the analysis of proteins in FFPE tissues. Incubation of tissue sections at high temperature with a novel extr ...[more]

PMID: 20604508

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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

FFPE - Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics

Publications

Formalin-fixed paraffin-embedded (FFPE) proteome analysis using gel-free and gel-based proteomics.

Similar Datasets

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