RNA was extracted using homogenization and Trizol reagent (Invitrogen, Carlsbad, USA) following the instructions from the manufacturer.
Label
Cy3 or Cy5
Label protocol
The mRNA (1–2 μg) was reverse-transcribed with random nonamer primers (1 μg per reaction) and T18VN (0.25 μg per reaction) to synthesize cDNA. The reaction contained a 1:1 mixture of 5-(3-aminoallyl) thymidine 5'-triphosphate (Sigma, St. Louis, USA) and thymidine triphosphate (TTP) in place of TTP alone. The cDNA products were bound to QIAquick PCR Purification columns (Qiagen, Hilden, Germany) following the manufacturer's instructions, washed three times with 80% ethanol, and eluted with water. Purified cDNA was reacted with N-hydroxy succinimide esters of Cy3 or Cy5 (Amersham Pharmacia Biotech, Piscataway, USA) following the manufacturer's instructions.
Hybridization protocol
Mixed labeled cDNAs were added to hybridization buffer containing 1 M NaCl, 0.5% sodium sarcosine, 50 mM methyl ethane sulfonate (MES), pH 6.5, 33% formamide and 40 μg herring sperm DNA (Invitrogen, Carlsbad, USA). Hybridizations were carried out in a final volume of 0.5 ml injecting into an Agilent hybridization chamber at 42°C on a rotating platform in a hybridization oven (Robbins Scientific Corporation, Sunnyvale, USA) for 16–24 h. Slides were then washed (rocking for approximately 30 seconds in 6 × SSPE, 0.005% sarcosine, then rocking for approximately 30 seconds in 0.06 × SSPE) and scanned with a 4000A microarray scanner (Axon Instruments, Union City, USA). Hybridizations were performed in duplicate with fluor reversal: that is, each mRNA sample was examined in duplicate, once in the Cy3 channel and once in the Cy5 channel, on separate arrays. Each array was hybridized with two samples simultaneously, each from an individual tissue.
Scan protocol
Slides were scanned with a 4000A microarray scanner (Axon Instruments, Union City, USA).
Description
Using a quantitative alternative splicing (AS) microarray platform, we have identified a large number of widely-expressed mouse genes containing single or coordinated-pairs of alternative exons that are spliced in a tissue-regulated fashion. The majority of these AS events display differential regulation in central nervous system (CNS) tissues. Approximately half of the corresponding genes have neural-specific functions and operate in common processes and interconnected pathways. Differential regulation of AS in the CNS tissues correlates strongly with a set of mostly new motifs that are predominantly located in the intron and constitutive exon sequences neighboring CNS-regulated alternative exons. Different subsets of these motifs are correlated with either increased inclusion or increased exclusion of alternative exons in CNS-tissues, relative to the other profiled tissues.
Data processing
TIFF images were quantitated with GenePix 3.0 (Axon Instruments). Individual channels were spatially denoised (i.e., overall correlations between spot intensity and position on the slide removed) by using spatial trend removal (STR) with 10% outliers. We applied variance stabilizing normalization (VSN) by using 25% of the genes to normalize all single channels to each other. Finally, we manually identified and removed data from residual artifacts apparent on microarray images and measurements that showed high inconsistency between dye-swaps.
