Phase 1 The Original Experimental Design

Our laboratory recently undertook a series of array-based experiments designed to identify cDNAs that were expressed in different primary cell isolates from the cardiovascular system. Our aim was to profile the complement of cDNAs expressed in cultured rat cardiomyocytes, cardiac fibroblasts, and aortic smooth muscle cells, all isolated from neonatal rats. We hoped to generate a list of marker genes that would make up a molecular phenotype for each of the cardiovascular cell types. Next, we planned to use this information to assay for any "transdifferentiation" events that might take place after overexpression of cardiac-specific transcription factors GATA4, MEF2C, and Nkx2.5 [7-9] in fibroblasts or smooth muscle cells. Furthermore, this approach was expected to identify novel endogenous targets of these transcription factors.

In order to have the most reliable data possible, we controlled for biological variation by independently preparing RNA from three separate primary cell isolations of each cell type. For each array hybridization we used fresh arrays purchased from ResGen. In this way we could control for differences in culture conditions from month to month, and from various lots of printed arrays. We used fresh arrays for every measurement because we had previously determined that ResGen arrays can not be reliably stripped and re-probed.

Our first set of array experiments were designed to establish a "baseline" of gene expression in each to the three cell types. Nine independent RNA isolations were performed, three from each of three cell types, neonatal cardiomyocytes, cardiac fibroblasts, and aortic smooth muscle cells.

We chose to employ microarrays containing 5147 cDNAs spotted onto nylon filters marketed by Invitrogen, Inc. and manufactured by ResGen, Inc. (Fig. 7.1). In the fall of 1999 we purchased a set of filters and the associated probe labeling reagents and hybridization buffers to expedite our studies. These filters were chosen for several reasons, including the low initial startup cost, the fact that the reagents and methods for probe preparation and array visualization were already present in our lab, and that at the time, ResGen had by far the largest commercially available collection of rat cDNAs present on an array. Over the course of a year, we performed a series of 16 hybridizations to the gf300 arrays and analyzed the output data with custom spreadsheets developed in our lab.

Total RNA was extracted from these three primary isolates of neonatal rat cardiovascular cells and used to generate probes for the arrays. Rat gf300 arrays were hybridized to 33P-labeled reverse transcribed probes and all arrays were scanned on a phosphorimager. The images were imported to Pathways 2.0 software package (ResGen, Inc.). This software was used to identify each of the cDNAs spotted onto the array and generate a report on the background and signal intensity of each spot. The raw data reports were exported into Microsoft Excel and processed through a series of calculations to subtract background, and normalize each signal to the average signal of the array. We next took the normalized array measurements from three independent hybridizations, averaged the signal intensities for each

Fig. 7.1 The gf300 array from Research Genetics. 5,123 individual cDNAs are spotted onto the 5 cmX7 cm nylon membrane. Each membrane is cut in the upper right corner for orientation. The cDNA spots are arranged in two fields, 1 and 2, each field containing 8 individual grids laid out right to left, A through H. Each grid is made up of individually spotted cDNA clones arranged in 12 col umns of 30 rows. Along with the 1,683 known genes and 3,440 ESTs present on the gf300 array, a series of controls are present including 228 total genomic DNA spots and 228 beta actin spots used for normalization of the signal and for orientation and alignment of the membrane when using the software from Research Genetics. The spacing between each spot is ~750 microns from center to center.

Fig. 7.1 The gf300 array from Research Genetics. 5,123 individual cDNAs are spotted onto the 5 cmX7 cm nylon membrane. Each membrane is cut in the upper right corner for orientation. The cDNA spots are arranged in two fields, 1 and 2, each field containing 8 individual grids laid out right to left, A through H. Each grid is made up of individually spotted cDNA clones arranged in 12 col umns of 30 rows. Along with the 1,683 known genes and 3,440 ESTs present on the gf300 array, a series of controls are present including 228 total genomic DNA spots and 228 beta actin spots used for normalization of the signal and for orientation and alignment of the membrane when using the software from Research Genetics. The spacing between each spot is ~750 microns from center to center.

cDNA and calculated the standard error of the mean. Differential expression between the RNA isolated from two cell types was determined by performing an unpaired Students T-test for each spot, and p<0.05 was taken as statistically significant. Each of the three cell types under study was analyzed in a pair-wise manner with the others.

We identified several cell-type specific cDNAs from our array analysis and published the results as an abstract at the 2000 meeting of the American Heart Association [10]. Many of the genes we identified as being differentially expressed in a cell-type specific manner agreed with previously published and well known cardiovascular cell markers. In RNA isolated from cardiomyocytes, we detected specific expression of transcripts that encode atrial natriuretic factor, desmin, and cardiac isoforms of troponin and myosin [11-14]. In smooth muscle cells, transcripts for the SM22 protein and osteopontin were identified, again agreeing with well described expression markers for this cell type [15, 16]. These results gave us confidence that the microarray methodology we were using was working as predicted, and that the additional novel transcripts we identified as cell-type specific were also correct. Interestingly, we had identified multiple ESTs specifically expressed in cardiomyocytes and aortic smooth muscle, and these transcripts were to be the focus of further study in our lab.

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