The Huck Institutes of the Life Sciences

Affymetrix ST microarray

Protocols for Affymetrix Whole Transcript Sense Target Labeling (ST) microarrays

  1. Accurately determine RNA concentration and purity using NanoDrop and Qubit.
  2. Assess total RNA quality using RNA Nano Chips on the Agilent Bioanalyzer. RINs should generally be 7-10 but a narrow range of scores is most important.
  3. Using 100 ng of total RNA from each sample, generate second-cycle single stranded sense strand cDNA using the Ambion WT Expression Kit (#4411974) according to the manufacturer’s protocol.
  4. Fragment the sense strand cDNA according to the manufacturer’s instructions using the Affymetrix GeneChip WT Terminal Labeling Kit (#900671) and confirm proper fragmentation using an RNA Nano Chip on the Agilent Bioanalyzer.
  5. Label the resulting fragmented cDNA using the Affymetrix GeneChip WT Terminal Labeling Kit (#900671) according to the manufacturer’s protocol. Assess labeling efficiency with the gel shift assay described by the manufacturer but utilizing an Agilent Bioanalyzer RNA Nano Chip instead of PAGE.
  6. Prepare hybridization cocktail using the GeneChip Hybridization Wash and Stain Kit (#702232) and hybridize arrays according to the manufacturer’s instructions in the Affymetrix GeneChip Hybridization Oven 640.
  7. Following hybridization; wash and stain arrays using the Affymetrix GeneChip Fluidics Station 450 according to the manufacturer’s protocol using the GeneChip Hybridization Wash and Stain Kit (#702232)
  8. Scan the stained arrays using the GeneChip Scanner 3000 7G.