Preparation of RNA samples

  Note:  The quality of the RNA is essential to the overall success of the GeneChip analysis.  Since the most appropriate protocol for the isolation of RNA can be source-dependent, we recommend using a protocol that has been established for the tissues and cells being used.  In the absence of an established protocol, we suggest using one of the commercially available kits designed for RNA isolation. 

  Either total RNA or purified poly(A) mRNA are acceptable.

Isolation of RNA from mammalian cells or tissues

  When using a commercial kit, follow the manufacturer's instructions for RNA isolation.  Good quality  RNA has been obtained using the kits listed below:

Total RNA preparation from cells:  QIAGEN's RNeasy Total RNA Isolation kit.

Total RNA preparation from tissues:  Invitrogen Life Technologies' TRIzol reagent.

Poly(A) mRNA isolation:  QIAGEN's Oligotex mRNA kit.

  Note:  When total RNA is isolated using TRIzol, a second RNA cleanup step using QIAGEN RNeasy Total RNA isolation kit is recommended.  This gives much better yield of labeled cRNA.

  It is not necessary to precipitate total RNA following isolation or cleanup with RNeasy columns. 

  A minimum of 5ug total RNA at 0.5-1.0 ug/ul concentration, or 0.2ug poly(A) mRNA at >0.02 ug/ul concentration will be needed to obtain sufficient quantity of labeled cRNA for target assessment and hybridization to GeneChip expression probe arrays.