Methods, Services and Prices (Version 1.9.2007)
DNA microarray analyses represent the current state-of-the-art technology for whole genome expression profiling and simultaneous detection of genome-wide transcriptional responses activated by individual or complex signals.
“Wet lab technology”. Usually the RNA is provided by the user, however, we offer to prepare RNA upon request. We require 3-5µg total RNA of high quality. Lower amounts (100-1000ng) can be processed upon request. For RNA preparation we recommend a combination of the “TriReagent” preparation method (Molecular Research Center, Cincinnati, OH) with the “RNeasy” protocol (Quiagen, Hilden, Germany). We routinely reassess RNA quantity and integrity by OD260/280 measurements and “Agilent lab-on-a-chip” technology (2100 Bioanalyzer, Agilent, Palo Alto, CA). Quality requirements are OD260/280 ratios of 1.8 to 2.2 and an 18S to 28S ratio of about 1.8 to 2.2.
Target labeling and hybridization: Hybridization target preparations are performed according to protocols recommended by Affymetrix (Affymetrix Technical Manual). Briefly, for 3’ end arrays (like the U133 series), 1.5µg total RNA is reverse transcribed into cDNA using an oligo(dT)-T7 promotor primer and transformed into double stranded cDNA by E.coli DNA polymerase using the Affymetrix one cycle cDNA synthesis kit. After purification of double stranded cDNA with the Affymetrix GeneChip Sample Cleanup Module, biotin-labeled cRNA is produced by T7 polymerase (Affymetrix IVT Labeling kit). After Agilent-based quantification and integrity control, 20 µg cRNA is fragmented by alkaline treatment (Affymetrix GeneChip Sample Cleanup Module) and 15 µg fragmented cRNA added to the hybridization cocktail (300 µl final volume). Details concerning target preparation for sense target arrays (like the Exon arrays) are provided upon request. The arrays are washed and stained according to the recommended Fluidics Station protocol (EukGE-WS2 version 5_450). Fluorescence signal intensities from each feature on the microarrays are determined using the Affymetrix GeneChip Scanner 3000 and the GCOS software (version 1.2) according to the manufacturer‘s recommendations.
Data processing and presentation. The raw data from all arrays of a given experiment are normalized using the RMA package for “R” by R. Irrizary (Biostat. 4: 249-264; 2003) for Exon-type arrays and with GC-RMA (Wu et al., http://www.bepress.com/jhubiostat/paper1/) for 3’ end arrays. The final data will be provided as expression values for each probe set (original Affymetrix cel.files and xls.files in the case of 3’ arrays) and one set of regulation tables (xls.files), if requested. Additional bioinformatic services are offered upon request or by the GDCF-Bioinformatics Unit (F. Überall).
MiRNA screening and promoter tiling arrays. MicroRNA arays and promoter tiling arrays are being analysed in our facility, however, this service is not being offered on a routine basis.
Prices:
The EPU is run as a non-profit organization and, in general, offers its services to members of academic institutions only on a cost reimbursement basis. Please inquire for details (Reinhard.Kofler@i-med.ac.at, +43-512- 570485-11).