Gene Expression Analysis Service

The Affymetrix GeneChip® product range for gene expression analysis enables researchers to qualitatively and quantitatively study the gene expression levels in several model organisms (see table 1 and Affymetrix for a complete list of catalog arrays). Alternatively the Custom Express ™ Array Program and NimbleExpress™ Array Program can be used to design GeneChip® expression arrays representing 520 to 61,000 sequences per array and study any completely sequenced genome or user-specific subset of genes.

High-quality RNA is an absolute prerequisite to obtain reliable and reproducible data. Based on our experiences with a variety of organisms ranging from yeast over mammalian tissues to Drosophila and Arabidopsis we provide you with advice on how to isolate RNA from your samples. For the standard protocol 1 µg of total RNA is needed, but, if tissue or cells are limited, a small sample protocol can be used that reduces this starting amount to as low as 50 ng of total RNA. The A260/A280 ratio should be between 1.8 and 2.1 for pure RNA. Upon receipt of the RNA samples Gene Express assesses their concentrations and A260/A280 ratios using a NanoDrop® ND-1000 spectrophotometer. The integrity of the RNA is analyzed using the RNA 6000 Nano Assay on an Agilent 2100 Bioanalyzer. If the resulting electropherogram (example) meets the quality parameters, Gene Express proceeds with the processing of the sample.

The Gene Expression Analysis Service follows this general workflow for the standard protocol (technology and graphical overview):

  • Advice on suitable experimental strategy, RNA extraction etc.
  • Quality check of RNA samples on Agilent 2100 Bioanalyzer (example)
  • First and second strand cDNA synthesis
  • In vitro transcription, resulting in biotinylated cRNA
  • Fragmentation of biotinylated cRNA
  • Quality check of non-fragmented and fragmented cRNA on Agilent 2100 Bioanalyzer (example non-fragmented and fragmented cRNA)
  • (Hybridization to Test3 Arrays, if indicated, e.g. after cRNA quality check)
  • Hybridization to GeneChip® array
  • Staining and washing of array
  • Scanning with GeneChip® scanner 3000
  • Quality check of raw data using Affymetrix software (GCOS 1.1)
  • Expression analysis (comparisons)
  • Higher-level analyses of data obtained (optional)

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