Almost indefinite numbers of formalin-fixed, paraffin-embedded tissue specimens (FFPE) are stored worldwide and for many of them rich clinical documentation is available. Reliable extraction of molecular information would allow understanding mechanisms of human pathology and disease.
The aim of the FFPE Profile project is to find the best way how to obtain the most accurate expression profiles from FFPE samples. We will find the best available solutions for
• RNA isolation
• cDNA synthesis and amplification
• labeling
• microarray platform and processing
• data analysis
• extraction of clinically relevant information
We will take two fresh frozen biopsies for which we also have large amounts of FFPE material. These two fresh frozen samples will be characterized in great detail using standard methods. Differential expression between these two fresh frozen samples will be used as the “true” differential expression.
Each of the evaluated methods will be performed in at least three laboratories to obtain “objective” information about their performance.
FFPE Profile – A project in milestones
FFPE Profile will be performed in individual milestones. For each milestone, one of the above described sample processing steps will be evaluated, and the results will be published.
Milestone 1: RNA isolation
We will find out which available chemistry provides highest quality and quantity of RNA from FFPE samples. RNA will be evaluated for quantity and integrity using capillary electrophoresis (Agilent, Bioanalyzer). RNAs from each of the isolation chemistries will be amplified by a single method and the amplified material will be analyzed on a single type of microarray. This allows seeing differences in performance of isolation chemistries.
Milestone 2: Amplification
RNA as isolated by the best performing isolation method will be subject to different amplification chemistries to obtain large amounts of cDNA. cDNAs from each of the amplification chemistries will be analyzed on a single type of microarray. This allows seeing differences in performance of amplification chemistries.
Milestone 3: Labeling and microarrays
cDNA as amplified by the best performing isolation method will be subject to labeling and microarray hybridization using different microarray platforms. The combination of labeling and the specific type of microarray will be decided by the microarray providers. Using the data analysis method recommended by the microarray provider, this allows seeing differences in performance of microarrays.
Milestone 4: Data analysis
Microarray raw data as generated by the best performing microarray platform will be analyzed by different algorithms and data analysis concepts. One could for example imagine that certain probes are specifically susceptible to problematic measurements in FFPE samples and therefore should be excluded. This allows seeing differences in performance of data analysis concepts.
Milestone 5: Extraction of clinically relevant information
During milestones 1 to 4 the optimal combination of RNA isolation, amplification, microarray platform and data analysis will have been determined and subsequently, this knowledge will be applied to a clinically relevant question. FFPE sample of breast cancer will be analyzed for expression profiles. Breast cancer is well characterized on the transcriptomic level and we expect to gain even deeper insight into molecular sub-classification of breast cancer since FFPE specimen provide rich clinical information and the newly developed expression profiling method will provide rich molecular information for those clinically well characterized samples.
Who can participate?
Whoever believes that he/she can contribute to a better understanding how to obtain expression profiles from FFPE samples should contact us (see later how). A contribution can be sample processing, data analysis or consumables. We already got positive feedback from several of the companies from whom we will use chemistry or arrays.
Timeline
Updates on conference calls and project time line will soon be available here.
Contact
Please contact us with the web form below or send us an email to herbert.auerirbbarcelona.org .
