Bone marrow total RNA was ordered from Clontech (Palo Alto, CA). Total RNA was extracted from 200-400 mg of frozen tissue using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was additionally purified with the RNAeasy kit (Qiagen, Valencia, CA). Labeled cDNA for array hybridizations was generated by linear amplification of total RNA, followed by direct labeling of the amplification products. Briefly, RNA samples from five normal tissues were individually amplified using the BD SMARTTM mRNA amplification kit (BD Biosciences, Palo Alto, CA). 1.5 ug of total RNA from each normal tissue was used as a substrate for amplification according to manufacturer’s specifications. Each amplified sample was run on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) to check the quality and integrity of the mRNA. Typically, the amplification products migrated as a broad peak, with the majority of transcripts ranging from 500 to 4000 bp. The amplified mRNAs were prepared for hybridization as follows: a pool of all the samples to be hybridized to the splicing arrays was assembled by mixing equal amounts of mRNA from each of the five tissue samples and four glioblastoma (GBM) samples (not discussed in this study). This comparator pool was used for all hybridizations. For each hybridization, 250 ng of the amplified normal tissue mRNA or GBM mRNA and 250 ng of the pool were labeled with Cyanine 5-dCTP and Cyanine 3-dCTP, respectively (Perkin Elmer, Boston, MA) using the Agilent Fluorescent Direct Label kit (Agilent Technologies, Palo Alto, CA). Dye-swapped labeling reactions were performed in parallel and hybridized to the second array on the slide. Labeling reactions were carried out according to the manufacturer’s specifications, with the following modification: instead of the DNA primer provided with the kit, 2 ug of random hexamers (MWG Biotech, High Point, NC) were used to prime the reaction. The entire Cyanine 5 and Cyanine 3 labeling reactions were combined according to the Fluorescent Direct Label kit protocol and were prepared for hybridization to the array using the In situ Hybridization kit Plus (Agilent Technologies, Palo Alto, CA) according to manufacturer’s specifications, with the exception that hybridizations were performed for 36-48 hrs. Slides were washed and dried according to manufacturer’s specifications and scanned on an Agilent Microarray Scanner (Model G2565BA; Agilent Technologies, Palo Alto, CA). Scanned images were analyzed using the Agilent Feature Extraction Software (Version A.7.1.1; Agilent Technologies, Palo Alto, CA). Keywords = Alternative Splicing Keywords = Agilent
