Microarray-Analysis - Workflow
Before starting the experiment, we offer advice on preanalytics and assist if necessary in the experiment design. The actual experiment according to the RNA / DNA preparation is divided into the following steps:
Workflow: Expression Analysis
- starting material: total-RNA or Poly-A-RNA
- recommended starting amounts: 50-500 ng RNA (concentration: min. 20 ng/µl)
- cDNA synthesis
- in vitro transcription (cRNA synthesis)
- fragmentation of cRNA
- hybridization onto the array (16h at 45ºC)
- washing and staining (these steps are largely automated and are performed with a Fluidics Station)
- scanning (Affymetrix GeneChip Scanner 7G)
At each level of preparation appropriate quality checks of the steps will be performed.
Workflow: Whole-Transcript Expression - GeneST-/ Exon-Arrays
- starting material: total-RNA
- recommended starting amounts: 50-500 ng RNA (concentration: min. 20 ng/µl)
- cDNA synthesis
- in vitro transcription (cRNA synthesis)
- 2nd cycle, 1st strand cDNA
- aRNA hydrolysis
- cleanup of sense strand DNA
- fragmentation
- terminal labeling
- hybridization on the Array (16h at 45ºC)
- washing and staining (these steps are largely automated and are performed with a Fluidics Station)
- scanning (Affymetrix GeneChip Scanner 7G)
At each level of preparation appropriate quality checks of the steps will be performed.
Workflow: SNP-Analysis
- starting material: genomic DNA
- starting amount: 500 ng DNA (concentration: min. 50 ng/µl)
- digestion (depending on the array-type: Xba I, Hind III, Nsp I, Sty I)
- adapter ligation
- PCR
- fragmentation
- labeling
- hybridization on the array (depending on the array-type: 16h at 48ºC oder 16h at 49ºC)
- washing and staining (these steps are largely automated and are performed with a Fluidics Station)
- scanning (Affymetrix GeneChip Scanner 7G)
At each level of preparation appropriate quality checks of the steps will be performed.
