Advances in the analysis of gene expression have revolutionized the ability to evaluate and understand the mechanisms of cancer and therapeutic response. The molecular understanding of disease has been greatly advanced by the use of modern mRNA profiling. Reports from several cancer studies have shown that DNA microarray technology allowed for the identification of many different subtypes of diseases (Perou et al. 2000, Golub et al. 1999, Bhattacharjee et al. 2001) and that these subtypes often have important clinical implications (Sørlie et al. 2001, Shipp et al. 2002). However, applying these methods into clinical studies has been limited due to its reliance on fresh tissue. The majority of expression profiling publications use high quality RNA from frozen samples. However these studies have been restricted due to the small number of samples in these collections. On the other hand, there is a huge resource of FFPE (formalin-fixed and paraffin embedded) tissues specimens held in histopathology departments. These samples provide an invaluable resource for retrospective studies correlating molecular features with therapeutic response and clinical outcome. However extraction of RNA from these tissues has proved to be problematic due to the detrimental effects of formalin-fixation (s. fig. Comparison of RNA Quality).
We have implemented Illumina’s Whole Genome cDNA-mediated Annealing, Selection, extension and Ligation assay (WG-DASL, Bibikova et al., 2004) to analyze more than 24.000 transcripts (Human Sentrix-8 BeadArray) in parallel, using RNA isolated from FFPE samples. For details see 'Principle of Whole Genome DASL'. The technical reproducibility that is achieved using WG-DASL is in the range of r2
YOU supply
- At least 8 samples or multiple thereof
- Amount of RNA >300-500 ng, to allow for input QC
- Concentration of >75 ng/µl
- RNA index >3; eaqual for all samples to be compared
- average RNA fragment size needs to be similar for all samples to be compared
- Select 'FFPE Human Sentrix-8' in the field for chip type in the submission form
- We recommend to run at least twice as many biological replicates as compared to classical expression profiling done by using intact RNA samples
What we do
- QC of input RNA (Bioanalyzer and Nanodrop)
- First strand cDNA synthesis (oligo dT and random priming)
- DASL procedure (details)
- Hybridization to human Sentrix-8 BeadArrays
- Image analysis
- Overall QC
Output to YOU
- All RNA and labeling QC data (conc. of input RNA, quality of input RNA, conc. of double stranded cDNA, conc. of labeled cRNA, quality of cRNA)
- Raw data of microarrays
- Individual chip QC (for chip integrity and hybridization success)
- Raw data evaluation (box plots)
- Interpreted data (numerical readout and gene annotation for microarray elements (probe sets)
- Normalization across each chip
- Normalization (if wanted) for complete sets of experiments
What's next?
You may unlock the insights buried in your experimental data, applying statistical analysis using DKFZ' in-house Chipster installation that makes use of R and Bioconductor packages and a Java GUI. Pathway analysis tools like Ingenuity Pathway Analysis or DAVID are also available via direct links, or provision of a computer workstation at GPCF (booking system for Ingenuity). Check out our webpage for more details and to get access.
For more Information, please review the FAQ site on expression profiling and the respective glossary.
Please consult the pricelist for detailed cost information.
