Biopsy cores were embedded individually in polyethylene glycol freezing media (Tissue-Tek OCT Compound, Sakura Finetek, Torrance, CA) and placed in isopentane that was precooled in liquid nitrogen. Specimens were then placed at 80°C until laser-capture microdissection (LCM). At time of LCM, 8-um frozen sections were obtained and immediately fixed in cold 95% ethanol. Sections were briefly (5 to 10 seconds) stained with hematoxylin using the Arcturus HistoGene Staining Solution (Arcturus Bioscience, Mountain View, CA) and dehydrated in 100% ethanol followed by xylenes (described in the Arcturus HistoGene LCM Frozen Section Staining Kit protocol). Five thousand epithelial cells from histologically benign glands were obtained by LCM using the Arcturus PixCell II instrument (modified from Emmert-Buck as described in Moore S, Knudsen B, True LD, et al: Loss of stearoyl-CoA desaturase expression is a frequent event in prostate carcinoma. Int J Cancer 114:563-571, 2005). Digital photos were taken of tissue sections before, during, and after LCM and assessed by an experienced genitourinary pathologist (B.S.K.) to confirm the histology of the laser captured cells. Each slide capture session lasted no longer than 20 minutes to minimize RNA degradation. The laser capture settings were 55mWbeam, 1.5 ms pulse, and 15um spot size. Cells were lysed in Arcturus RNA Extraction Buffer, RNA was isolated using the Arcturus PicoPure RNA Isolation Kit, and the samples were treated with DNAse using the Qiagen RNase-Free DNAse Set (Qiagen Inc, Valencia, CA). RNA was amplified for two rounds using the Ambion MessageAmp aRNA Kit (Ambion Inc, Austin, TX), and sample quality and quantity were assessed by agarose gel electrophoresis and absorbance at A260. To provide a reference standard RNA for use on cDNA microarrays, we isolated total RNA from LNCaP, DU145, PC3, and CWR22 cell lines (American Type Culture Collection, Manassas, VA) growing at log phase in dye-free RPMI-1640-1640 medium supplemented with 10% fetal bovine serum (FBS; Life Technologies, Rockville, MD). RNA was purified using Trizol (Life Technologies) following the manufacturer’s protocol.
Label
Cy5
Label protocol
We prepared and hybridized spotted cDNA microarrays as previously described (Moore S, Knudsen B, True LD, et al: Loss of stearoyl-CoA desaturase expression is a frequent event in prostate carcinoma. Int J Cancer 114:563- 571, 2005) using RNA from a single batch of reference standard for each hybridization.
Human Prostate 030020_BI. Specimen_ID: 030020_BI; Age (years): 67; Race/Ethnicity: White; PSA (ng/dL): 5.6; Total Operative Time (minutes) : 180; Treatment: None; Gleason Sum: 7; pStage: T3aN0; Prostate Volume (mL): 43; Nerve Sparing: None; Patient Number: 1; Type: Needle; Ischemia Time (minutes): 36; Estimated Blood Loss (mL): "1,100";
Extracted molecule
total RNA
Extraction protocol
Biopsy cores were embedded individually in polyethylene glycol freezing media (Tissue-Tek OCT Compound, Sakura Finetek, Torrance, CA) and placed in isopentane that was precooled in liquid nitrogen. Specimens were then placed at 80°C until laser-capture microdissection (LCM). At time of LCM, 8-um frozen sections were obtained and immediately fixed in cold 95% ethanol. Sections were briefly (5 to 10 seconds) stained with hematoxylin using the Arcturus HistoGene Staining Solution (Arcturus Bioscience, Mountain View, CA) and dehydrated in 100% ethanol followed by xylenes (described in the Arcturus HistoGene LCM Frozen Section Staining Kit protocol). Five thousand epithelial cells from histologically benign glands were obtained by LCM using the Arcturus PixCell II instrument (modified from Emmert-Buck as described in Moore S, Knudsen B, True LD, et al: Loss of stearoyl-CoA desaturase expression is a frequent event in prostate carcinoma. Int J Cancer 114:563-571, 2005). Digital photos were taken of tissue sections before, during, and after LCM and assessed by an experienced genitourinary pathologist (B.S.K.) to confirm the histology of the laser captured cells. Each slide capture session lasted no longer than 20 minutes to minimize RNA degradation. The laser capture settings were 55mWbeam, 1.5 ms pulse, and 15um spot size. Cells were lysed in Arcturus RNA Extraction Buffer, RNA was isolated using the Arcturus PicoPure RNA Isolation Kit, and the samples were treated with DNAse using the Qiagen RNase-Free DNAse Set (Qiagen Inc, Valencia, CA). RNA was amplified for two rounds using the Ambion MessageAmp aRNA Kit (Ambion Inc, Austin, TX), and sample quality and quantity were assessed by agarose gel electrophoresis and absorbance at A260. To provide a reference standard RNA for use on cDNA microarrays, we isolated total RNA from LNCaP, DU145, PC3, and CWR22 cell lines (American Type Culture Collection, Manassas, VA) growing at log phase in dye-free RPMI-1640-1640 medium supplemented with 10% fetal bovine serum (FBS; Life Technologies, Rockville, MD). RNA was purified using Trizol (Life Technologies) following the manufacturer’s protocol.
Label
Cy3
Label protocol
We prepared and hybridized spotted cDNA microarrays as previously described (Moore S, Knudsen B, True LD, et al: Loss of stearoyl-CoA desaturase expression is a frequent event in prostate carcinoma. Int J Cancer 114:563- 571, 2005) using RNA from a single batch of reference standard for each hybridization.
Hybridization protocol
We prepared and hybridized spotted cDNA microarrays as previously described (Moore S, Knudsen B, True LD, et al: Loss of stearoyl-CoA desaturase expression is a frequent event in prostate carcinoma. Int J Cancer 114:563- 571, 2005) using RNA from a single batch of reference standard for each hybridization.
Scan protocol
Fluorescence array images were collected for both Cy3 and Cy5 using a GenePix 4000B fluorescent scanner (Axon Instruments, Foster City, CA), and GenePix Pro 4.1 software was used to grid and extract image intensity data.
Description
Human Prostate 030020_BI from Human hybridized against Reference
Data processing
Spots of poor quality, as determined by visual inspection, were removed from further analysis. To normalize log-ratio data, a print-tip specific Lowess curve was fit to the log-intensity versus log-ratio plot, using 20.0% of the data to calculate the fit at each point. This curve was used to center the log-ratio for each spot. Data were filtered to exclude poorly hybridized cDNAs by removing values with average foreground-minus-background intensity levels less than 300. We used the average of the two duplicatecDNAsspotsoneach microarray chip in subsequent analyses. Data were filtered to include clones returning data for at least 75% of the samples in both presurgery and postsurgery groups, which reduced the initial list of 6,751 clones to 5,753 clones.
