age: 36 gender: male ejection fraction: 64 left ventricular end diastolic diameter: 50 inflammation/PVB19: negative
Treatment protocol
endomyocardial biopsy from the right ventricle
Extracted molecule
total RNA
Extraction protocol
standard protocol TRIzol (Invitrogen)
Label
biotin labeled
Label protocol
Starting with amounts below 1 µg, cRNA targets were generated using the small sample protocol (Affymetrix, vers. II). Briefly, in vitro transcribed unlabeled cRNA from the first round of amplification was subjected to a second round of amplification. cRNA was transcribed into cDNA using random primers. After RNAse H mediated removal of surplus cRNA a second strand was synthesized using a T7 primer. In detail, total RNA amounts of 50-200ng were used for first strand synthesis. cDNA was synthesized starting with the annealing to 5pmol/ul of a T7- (dT)24 primer that contains a promotor for DNA dependent RNA polymerase (TIB Molbiol, Berlin, Germany) at 70°C for 6 min. Reverse transcription (RT) was performed at 42°C for 1 hr in first strand buffer containing 50mM TRIS-HCl (pH=8.3), 75mM KCl, 3mM MgCl2, 10 mM dithiothreitol, 500 µM each dATP, dCTP, dGTP, and dTTP, and 20,000 U/ml of Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). Second-strand synthesis was carried out in 20-µl solutions, using the complete cDNA reaction mix and the MgCl2-, dNTP and enzyme solutions of the two cycle synthesis kit (Affymetrix) The mixture was incubated at 16°C for 2 hours, followed by a further incubation at 75°C for 10 minutes. Double strand cDNA was directly subjected to the first round of amplification using the MEGAScript T7 Kit (Ambion). Incubation was carried out at 37°C for 16hrs The amplified cRNA was purified with an affinity resin column (RNeasy, Qiagen, Hilden, Germany). 50-600ng of the yielded cRNA was used for the second round. For first strand synthesis random primers (final concentration 0.2 µg/µl) were used. After incubation at 42°C for 1h RNAse H was added at 37°C for 20min.and the reaction was stopped by heating at 95°C. After addition of 5pmol/ul of a T7- (dT)24-primer, the mix was heated at 70°C for 6 min. Then the second strand reaction mix was added. The mixture was incubated at 16°C for 2 h followed by a further incubation with 2 µl of T4 DNA polymerase (5 U/µl) (Invitrogen, Karlsruhe, Germany) for 10 min. The reaction was terminated by addition of 10 µl of 0.5 M EDTA. Cleanup of double stranded cDNA was performed using the cDNA-clean-up-modul of Affymetrix. Synthesis of biotin-labeled cRNA was performed using the GeneChip IVT labeling kit (Affymetrix, Santa Clara, CA). The amplified cRNA was purified with an affinity resin column (RNeasy, Qiagen, Hilden, Germany) and fragmented by incubation at 94°C for 35 min. cRNA amounts were determined by UV-spectroscopy and distribution of cRNA fragment sizes of both, cRNA and fragmentation products, was checked by analyzing the samples on a LabChip (BioAnalyzer, AGILENT Technologies, Santa Clara, CA).
Hybridization protocol
After cleanup (Qiagen, Hilden, Germany), the biotin-labeled cRNA was fragmented by alkaline treatment (40 mmol/L Trisacetate (pH 8.2), 100 mmol//L potassium acetate, and 50 mmol//L magnesium acetate) at 94°C for 35 minutes. 15 µg of each cRNA sample was hybridized for 16 hours at 45°C to an Affymetrix Human Genome U133 Plus 2.0 GeneChip Array. Chips were washed and stained with streptavidin-phycoerythrin using a fluidics station according to protocolsrecommended by Affymetrix.
