laser-capture microdissected stroma derived from tissue sections of 10-weeks old mice bearing prostate intraepithelial neoplasia (PIN) - pool of 4 samples
LCM slides were prepared from serial 8-μm-thick frozen tissues sections placed on a polyvinyl nuclease free membrane (Molecular Machines&Industries, Glattbrugg, CH). Tissue sections were fixed in ethanol 70% (30 sec), stained with haematoxylin and eosin (15 sec each), dehydrated in graded ethanol, treated with xylene and air-dried in a sterile laminar flow hood. Slides were microdissected immediately following staining using a μCut Laser Microdissection system (Nikon Eclipse TE200). All steps and solutions were performed under RNase free conditions.
Growth protocol
standard mice maintenance in a specific pathogen-free facility according to Swiss guidelines for animal experimentation (authorization #1477)
Extracted molecule
total RNA
Extraction protocol
PicoPure RNA isolation kit (Arcturus, Mountain View, CA, www.arctur.com).
Label
Cy3
Label protocol
Labeled cDNA was obtained by reverse transcription of 5 μg of aRNA and incorporation of Cy3-dCTP and Cy5-dCTP (Amersham Biosciences, Amersham, UK).
Channel 2
Source name
laser-capture microdissected stroma derived from from tissue sections of 24-weeks old mice bearing invasive prostate tumor (PT) - sample 4
Strain: FVB. Gender: male. Age: 24-weeks old Tissue: prostate Tumor stage: invasive cancer stage (PT)
Biomaterial provider
Stamenkovic laboratory
Treatment protocol
LCM slides were prepared from serial 8-μm-thick frozen tissues sections placed on a polyvinyl nuclease free membrane (Molecular Machines&Industries, Glattbrugg, CH). Tissue sections were fixed in ethanol 70% (30 sec), stained with haematoxylin and eosin (15 sec each), dehydrated in graded ethanol, treated with xylene and air-dried in a sterile laminar flow hood. Slides were microdissected immediately following staining using a μCut Laser Microdissection system (Nikon Eclipse TE200). All steps and solutions were performed under RNase free conditions.
Growth protocol
standard mice maintenance in a specific pathogen-free facility according to Swiss guidelines for animal experimentation (authorization #1477).
Extracted molecule
total RNA
Extraction protocol
PicoPure RNA isolation kit (Arcturus, Mountain View, CA, www.arctur.com).
Label
Cy5
Label protocol
Labeled cDNA was obtained by reverse transcription of 5 μg of aRNA and incorporation of Cy3-dCTP and Cy5-dCTP (Amersham Biosciences, Amersham, UK).
Hybridization protocol
Hybridization of labeled cDNA to microarrays was performed for 16 h at 64 °C in a humidified chamber (Corning Costar, Cambridge, MA).
Scan protocol
Microarrays were imaged using the ScanArray 4000 scanner (Perkin Elmer, Foster City, CA); Cy3 and Cy5 fluorescence intensities were extracted using the ScanAlyze software (http://rana.lbl.gov/EisenSoftware.htm).
Description
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Data processing
Gene expression was quantified with the SMA package using print tip group lowess normalization without background subtraction. For each array and each clone log2 ratios (M values) and the average log2 intensities (A value) of Cy3 and Cy5 signals were thus obtained.
