Quality management coordinator:
Dr. Holger Sültmann
German Cancer Research Center (DKFZ)
Div. of Molecular Genome Analysis
Phone: + 49 6221 424705
E-Mail: h.sueltmann@dkfz.de
Quality Management "Microarrays"
The working group „Microarrays“ representing all microarray platforms in the NGFN specified strict quality criteria.
“We ensure the good quality of DNA and RNA specimens by performing complete checks. For example, we test the concentration and the purity of the material with spectrophotometric analyses,” explains Dr. Holger Sültmann, spokesman of the working group. “The quality of each individual microarray is carefully checked and documented following a standardized procedure.”
Specific quality assurance measures are:
- Specific storage and sample preparation methods.
The tissue for microarrays must be frozen or submerged in RNase inhibiting substances. - RNA isolation.
Standardized protocol for purification of RNA for microarray analyses. - RNA quality control.
Test by UV absorbance spectra, capillary electrophoresis, quantitative RT-PCR. - Feature production and quality control.
All PCR products are examined by gel electrophoresis for presence of bands and absence of double bands.
Each microtitre plate is labelled with a barcode and each processing step is stored in appropriate databases (e.g. iChip, QuickLIMS). Thus, all steps of the production process (from clone replication and PCR to preparation of 384 well spotting plates and its usage) can be tracked. - Microarray processing.
The quality is tested by optical parameters:
- scanning of at least 10% of microarrays from each batch after spotting
- scanning of every microarray after post-spotting treatment
- Syto 61 staining of selected microarrays from each batch - Hybridization.Hybridization is performed with known reference RNAs using automated hybridization machines.
- Data analysis.
Documentation and storage of data in an integrated database. The standards are exchanged on the platform and are available for collaboration partners in the networks.
Protocols
Many work steps to process microarrays have already been standardized and documented. This set of protocols was covers all different practical issues of microarray experiment like tissue sampling, RNA preparation, RNA amplification, oligonucleotide and cDNA microarray production, microarray hybridization and aspects of quality control, as well as useful proposals for experimental design and data analysis. These standards improve the quality of the results and ensure the transparency of experimental procedures. They are intended to be used by as many partners as possible in the NGFN on the basis of voluntary self-commitment.
Clinical cooperation partners of microarray platforms find important informations for optimized sample preparation and will get an overview of all experimental procedures. All protocols will be regularly updated. The feedback of protocol users will be continuously implemented. A new protocol version number will point to these changes and detailed information will be listed at the end of every protocol sheet.
The protocols are in pdf format and can be downloaded here.
Please note that due to technological improvements, the protocols may be subject to changes.
1. RNA Sample Preparation
1.1. Biopsy specimen collection and storage for RNA preparation
1.2. RNA extraction
1.1.2. RNA extraction from blood
1.2.2. Biopsy homogenization by dismembrator
1.2.3. RNA extraction very small samples
1.2.4. RNA extraction large samples
1.3. RNA quantification and quality control
1.3.1. RNA quality and quantity control
1.3.2. RNA concentration by precipitation
1.4. RNA amplification by T7 RNA Polymerase
1.5. RNA fluorescent_labeling
1.6. RNA common reference
2. cDNA Arrays
2.1. cDNA clone sets
2.1.1. ENSEMBL cDNA clone set human and mouse
2.1.2. UNIGENE3-1 cDNA clone set human
2.1.3. PCR product generation of cDNA probes
2.2. Array Fabrication
2.2.1. Fabrication of DNA microarrays - Biorobotic Microgrid II
2.2.2. Fabrication of DNA microarrays - Versarray Biorad
2.2.3. Spotting pins in performance tests
2.2.4. Post-Treatment of spotted cDNA-Arrays
2.3. Array Hybridization
2.3.1. cDNA Array Hybridization in Corning chamber
2.3.2. cDNA Array hybridization by Ventana Discovery Machine
2.4. Array Quality Control
2.4.1. cDNA microarray quality control by primer hybridization
2.4.2. Cyto61 control staining of cDNA microarrays
3. Oligonucleotide Arrays
3.1. 70mer Oligonucleotide Microarray Operon Set
3.2. ROC Affymetrix Expression Analysis
4. Data Management and Analysis
4.1 Experimental design
4.2 Classification
4.3 Recommendations for normalization of microarray data
List of authors involved in the development of standard protocols:
- German Cancer Research Center (DKFZ), Molecular Genome Analysis
Dr. Tim Beißbarth, Dr. Ruprecht Kuner, Marcus Ruschhaupt, Dr. Holger Sültmann (Disease gene expression profiling using microarrays,
Computational methods for functional genomics) - German Cancer Research Center (DKFZ), Division of Molecular Genetics
Dr. Meinhard Hahn (Senior Researcher: RNA-Expression Profiling Using DNA-Microarrays) - German Cancer Research Center (DKFZ), Intelligent Bioinformatics Systems
Dr. Benedikt Brors, Chris Lawerenz - Institut für medizinische Informationsverarbeitung, Biometrie und Epidemiologie at the LMU München
Prof. Dr. Ulrich Mansmann, Mar - Max-Planck Institute for Molecular Genomics, Automation Group
Dr. James Adjaye, Nicole Greiner, Dr. Ingo Fritz, Dr. Claus Hultschig, Thomas Nitsche, Thomas Przewieslik, Elisabeth Socha - Resource Center and Primary Database (RZPD), Berlin
Dr. Florian Wagner - Universitätsklinikum Heidelberg, Innere Medizin III, Otto-Meyerhof-Zentrum
PD Dr. Dieter Weichenhan
Websites of SMPs and Disease-oriented Genome Networks involved in this quality management project:
