Experimental procedures

Adult heart tissue was taken out of the animal, briefly rinsed in ice-cold PBS, and the ventricles were snap frozen in liquid nitrogen as soon as possible. Embryonic tissue was dissected in ice-cold PBS, and whole hearts frozen on dry ice immediately after dissection. Hearts were homogenized using a Brinkman Polytron homogenizer for adult tissue, and a hand-held glass homogenizer for embryonic hearts.

Total RNA was isolated using the Trizol reagent following the instructions of the manufacturer. Isolated RNA was further purified by a phenol-chloroform extraction, followed by ethanol precipitation. The quality and integrity of the isolated RNA was monitored by gel electrophoresis and by photometric determination of the ratio of OD260 to OD280. RNA that showed clear bands of 18S and 28S RNA on the gel, and an OD260/280 between 1.7 and 2.0 was used for probe synthesis.

Generation of biotinylated cRNA probe for the Affymetrix arrays was performed according to Affymetrix’s protocols. Briefly, first-strand cDNA was synthesized using a hybrid reverse transcription primer consisting of oligo-dT and T7 RNA polymerase promoter sequences. The single-stranded cDNA was then converted to double-stranded cDNA. cDNA corresponding to 6-8 micrograms of total RNA was then used for in vitro transcription using T7 RNA polymerase and two biotinylated nucleotide precursors. The resulting biotinylated cRNA was then fragmented to a size of approximately 50bp. GeneChips were hybridized over night with 20 micrograms of biotinylated cRNA probe. GeneChips were washed in Affymetrix Fluidics station using the standard protocol. Bound cRNA was visualized by binding of streptavidin/phycoerythrin conjugates to the hybridized GeneChip, followed by laser scanning of bound phycoerythrin.

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Experimental Design

The experimental strategy used for expression profiling of murine models is tailored to the phenotype of the individual model system under study. Age and time points were chosen to reflect the onset, progression, and late stages of the disease, based on published results or our own analysis.

Each experiment is performed in triplicates, unless the degree of observed biological variation requires a higher number of samples in order to achieve statistical significant differences between groups or time points.

For adult mice, probes are synthesized from total RNA extracted from single heart ventricles. This approach allows us to account for biological variation, e.g. to differentiate between changes in expression that relate to differences in between individual mice from those that are related to the disease process. For embryonic samples, 3-6 hearts derived from 1-2 litters were pooled for each probe, depending on the embryonic stage being analyzed.

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Page last modified: 07-Aug-2003