The Huck Institutes of the Life Sciences

Affymetrix 3' IVT microarray

Protocols for Affymetrix 3' IVT 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. Label 100 ng of each sample using the Affymetrix IVT Express Kit (#901228) according to the manufacturer’s protocol. Normal aRNA yield is approximately 60 ug with a range of 40-100 ug.
  4. Fragment aRNA according to the manufacturer’s instructions and confirm proper fragmentation using RNA Nano Chips on the Agilent Bioanalyzer.
  5. 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.
  6. 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)
  7. Scan the stained arrays using the GeneChip Scanner 3000 7G.