ABSTRACT: Titin is a striated muscle-specific giant elastic protein and largely responsible for the generation of the diastolic force in the cardiac myocyte. Cardiac titin undergoes developmental changes in isoform expression during the course of cardiac development. At present, at least five size classes of titin isoforms (N2B and N2BA-A1, A2, N1, N2) have been identified using SDS agarose gel electrophoresis. The larger titin isoform N2BAs gradually decreased with ages, in contrast, the smaller titin isoform N2B increased in normal cardiomyocyte, and cardiac myocytes containing a higher proportion of the smaller titin isoform N2B have stronger passive tension than that with a lower proportion of the larger titin isoform N2BAs. Recently we found an autosomal dominant mutation which caused totally opposite cardiac titin isoform expression as compared to developmental stages. The larger total titin isoform N2BA increased in mutant rat cardiac myocytes with ages instead of the smaller cardiac titin isoform N2B. For the moment, mechanism of titin isoform switch is still unknown, therefore, the mutant rats will give us nevol sight to elucidate the titin splicing mechanism. Experiment Overall Design: Total RNA was extracted using TRIzol according to manufacturer instruction and further purified by the RNeasy mini kit (Qiagen, Valencia, CA). Double stranded cDNA was synthesized from total RNA (SuperScript II system; Invitrogen). An in vitro transcription reaction was then performed to obtain biotin-labeled cRNA from the double-stranded cDNA (Enzo BioArray High Yield RNA Transcript Labeling kit; Enzo Diagnostics, Farmingdale, NY). The cRNA was fragmented before hybridization, and then mixed in a hybridization mixture containing probe array controls, BSA, and herring sperm DNA. A cleanup procedure was performed on the hybridization cocktail using an RNeasy spin column (Qiagen), after which it was applied to the Affymetrix Rat 230 2.0 probe array. Total eighteen hybridization experiments were performed in which each stage (1-day, 20-day and 49-day) was represented by three normal and three mutant individual ventricular RNA extracts. Hybridization was allowed to continue for 16 h at 45°C in a GeneChip 640 hybridization oven, after which the arrays were washed and stained with phycoerythrin-conjugated streptavidin (Molecular Probes, Eugene, OR). Images were scanned using a GeneArray scanner (Agilent Technologies, Palo Alto, CA) and GeneChip cel files were subsequently processed by the log scale robust multi-array analysis (RMA). The log scale robust multi-array analysis (RMA) estimates are based upon a robust average of log2 (B (PM)), where B (PM) are background corrected perfect match intensities. three replicates were available for these conditions: three wild types and three homozygotes for 1-day, three wild types and three homozygotes for 20-day, and three wild types and three homozygotes for 49-day.
