<p> Bauer Core Standard Protocol Title: Fragmentation of cRNA for Affymetrix GeneChips Pages: 2 Revision: Date: August 26, 2003 Author(s): Jennifer A. Couget Reviewers: Shufen Meng Contact: [email protected] Comment:</p><p>1. Purpose This protocol describes the fragmentation procedure of cRNA for Target Cocktail Hybridization of Affymetrix GeneChips.</p><p>2. Materials 5X Fragmentation Buffer (stored at room temperature) DEPC-water Labeled cRNA 0.65 ml RNase-free tubes to prevent evaporation</p><p>3. Instrumentation To maintain proper fragmentation temperature, using a Thermal Cycler is recommended, but not required.</p><p>4. Reagent preparation 5X Fragmentation Buffer may be bought as part of the Affymetrix GeneChip Clean-up Module (Affymetrix, p/n 900371)</p><p>5X Fragmentation Buffer</p><p>200mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc</p><p>1M Tris acetate pH 8.1 4 ml MgOAc 0.64 g KOAc 0.98 g DEPC-water to 20 ml</p><p>Mix thoroughly and filter through a 0.2 m vacuum filter. Aliquot and store at room temperature. 5. Procedure</p><p>5.1 It is usually best to perform the fragmentation on the same day as hybridization whenever possible to minimize freeze-thawing effects on cRNA. </p><p>5.2 Quantify cRNA concentration and A260/A280 ratio. cRNA must be at a minimum concentration of 0.6 µg/µl. If it is not, concentrate under vacuum without heat. A260/A280 ratio should be between 1.9 and 2.1.</p><p>5.3 Use 2 µl of 5X Fragmentation Buffer for every 8 µl of cRNA plus water together (ratio is 2:8). In addition, the cRNA final concentration in the fragmentation reaction must be no less than 0.5 µg/µl.</p><p>5.4 We recommend fragmenting 20 µg of cRNA in a 40-µl fragmentation reaction.</p><p>5.5 For example, cRNA with a concentration of 1 µg/µl:</p><p> cRNA (1 µg/µl) 20 µl Final concentration 0.5µg/µl DEPC-water 12 µl 5X Fragmentation Buffer 8 µl Final concentration 1X Total Volume 40 µl</p><p>5X Fragmentation Buffer 8 µl: cRNA plus water 32µl ratio is 2:8</p><p>5.6 Incubate at 94C for 35 minutes. Cool to 4C and place on ice.</p><p>5.7 Check quality of cRNA by Agilent Bioanalyzer using mRNA Smear Nano Assay.</p><p>5.8 Run 100 ng of unfragmented cRNA side-by-side with 1 µl fragmented undiluted cRNA directly from fragmentation reaction.</p><p>5.9 Assay should reveal long unfragmented cRNAs, majority greater than 400 bp and fragmented cRNAs ideally between 35 and 200 bp.</p><p>5.10 Use all remaining fragmented cRNA in target hybridization cocktail immediately.</p><p>5.11 Store fragmented cRNA at –20C short-term for immediate future hybridization.</p><p>5.12 Store unfragmented cRNA and extra fragmentation reactions at -80C long-term.</p>