The isoproterenol application (4 mg *kg -1 *d -1 ) was done via osmotic minipump over two weeks
Growth protocol
Mice were kept under conventional conditions and had access to tap water and rodent chow diet ad libitum.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated form heart tissue using the TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacture’s instruction. Additionally, a cleanup step with the RNeasy Kit (Qiagen GmbH, Hilden, Germany) was performed. The integrity of total RNA was checked by capillary electrophoresis (2100 Bioanalyzer, Agilent Technologies, Inc. Palo Alto, CA, U.S.A.).
Label
Cy3
Label protocol
Labelled cDNA probes were synthesized from 20 µg of total RNA. Reverse transcription was carried out in a 30 µl reaction volume with 0.5 µg oligo-dT18-primer, 10 mM DTT, 20 U RNaseOut, 200 µM of each (dATP, dCTP, dGTP), 80 µM dTTP, 40 µM Cy3-/Cy5-dUTP (Amersham Pharmacia Biosciences, Freiburg, Germany), 200 U of Superscript III (Invitrogen, Karlsruhe, Germany). The labelled cDNA was purified using QIAquick PCR purification columns (Qiagen GmbH, Hilden, Germany) according to manufacturers protocol.
The isoproterenol application (4 mg *kg -1 *d -1 ) was done via osmotic minipump over two weeks
Growth protocol
Mice were kept under conventional conditions and had access to tap water and rodent chow diet ad libitum.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated form heart tissue using the TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacture’s instruction. Additionally, a cleanup step with the RNeasy Kit (Qiagen GmbH, Hilden, Germany) was performed. The integrity of total RNA was checked by capillary electrophoresis (2100 Bioanalyzer, Agilent Technologies, Inc. Palo Alto, CA, U.S.A.).
Label
Cy5
Label protocol
Labelled cDNA probes were synthesized from 20 µg of total RNA. Reverse transcription was carried out in a 30 µl reaction volume with 0.5 µg oligo-dT18-primer, 10 mM DTT, 20 U RNaseOut, 200 µM of each (dATP, dCTP, dGTP), 80 µM dTTP, 40 µM Cy3-/Cy5-dUTP (Amersham Pharmacia Biosciences, Freiburg, Germany), 200 U of Superscript III (Invitrogen, Karlsruhe, Germany). The labelled cDNA was purified using QIAquick PCR purification columns (Qiagen GmbH, Hilden, Germany) according to manufacturers protocol.
Hybridization protocol
Hybridization was done in a humidity chamber at 42°C for 16 h. Hybridization buffer contains 50% FA, 5xSSC, 0.1%SDS.
Scan protocol
The GenePix 4000B laser scanner (Axon Inc., USA) was used for visualising fluorescence signals and the GenePix Pro software (V 6.0) was used to calculate fluorescence intensities.
Description
none
Data processing
Raw data were intensity dependent normalized using the lowess function in GenePix Acuity4.0
