Laboratory of the Department of Oncology, 5405 University of Copenhagen at Herlev Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark
Treatment protocol
Plasmid DNA and in vivo Electro gene transfer: Five micrograms of the plasmid phGFP-S65T, encoding the green fluorescent protein (GFP), plasmid was dissolved in 20 μl PBS and injected intramuscularly along the fibres into the tibialis cranialis muscle of anaesthetised C57Bl/6 mice using an insulin syringe. Plate electrodes with 4-mm gap were fitted around the hind legs and an electric field was applied. The muscle was pulsed with a combination of a high voltage pulse (100 μsec, 1000V/cm) followed by a long low voltage pulse (400 ms, 100V/cm) with 1s lag between the pulses. The tibialis cranialis muscle, exercised 48 hrs after plasmid DNA and in vivo electro gene transfer.
Growth protocol
Six to eight weeks old female C57Black/C mice (average weight 22 g, Taconic, Denmark) were kept under pathogen-free conditions at 22 °C in a 14/10 hrs light/dark cycle with food and water ad libitum. All animal experiments were conducted in accordance with the recommendations of the European Convention for the Protection of Vertebrate Animals used for Experimentation and after permission from the Danish Animal Welfare Committee.
Extracted molecule
polyA RNA
Extraction protocol
Four hrs, 48 hrs and 3 weeks after treatment the mice were euthanized and the muscles were removed and placed in 1 ml solution D (guandinium thiocyanat, sodium citrate, sarcosyl and mercaptoethanol) on ice. Muscle extracts were prepared by homogenising the muscles with a rotor-stator homogeniser, and RNA were extracted using the Chomczynski and Sacchi method and digested with DNase. Due to low yield of total RNA, mRNA were amplified yielding antisense RNA (aRNA) using the MessageAMP II aRNA kit (Ambion, Europe).
Label
Biotin
Label protocol
5 μg aRNA was used to synthesize double stranded cDNA using Superscript® Choice System (Invitrogen, Denmark) with an oligo(dT) primer containing a T7 RNA polymerase promoter (GenSet, Evry, France). The cDNA was used as template for an IVT reaction to generate biotinlabelled antisense cRNA (BioArrayTM High Yield RNA Transcript Labelling Kit; Enzo Diagnostics, Farmingdale, NY, USA).
Hybridization protocol
The biotinlabelled antisense cRNA was fragmentated at 94°C for 35 min in fragmentation buffer (40 mM Tris, 30 mM MgOAc,10 mM KOAc), the labelled cRNA were hybridised for 16 hrs to Affymetrix MOE 430 2.0 arrays (Affymetrix, Santa Clara, CA, USA).The arrays were washed and stained with phycoerythrin conjugated streptavidin using the Affymetrix Fluidics Station® 400.
Scan protocol
The arrays were scanned in the Affymetrix GeneArray® scanner to generate fluorescent images, as described in the Affymetrix GeneChip protocol.
Description
Nothing in line.
Data processing
The cel image files (Affymetrix) were imported, pre-processed and analysed in DNA-Chip Analyser 2006 (http://www.dchip.org). The array files were normalised using the non-linear invariant set normalisation
method, choosing array (DNA-EP-48HRS) as baseline. Normalisation curves were visually inspected and none of the arrays were critical. In order to interpret the probe signal, model-based gene expression indexes
(MBEI) gene expression modelling was calculated using the PM/MM perfect match/ miss match) method. During the analysis array outliers were detected. None of the arrays exceeded 1.4% of array outliers
and the probe presence call was between 36.6% and 50.3%.
