|
| Status |
Public on Jul 14, 2006 |
| Title |
Prostate_03-155C_GL4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Prostate_normal_adjacent_cancer_03-155C
|
| Organism |
Homo sapiens |
| Characteristics |
Prostate normal adjacent from cancer patient 03-155C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following LCM, captured cells were lysed in RNA extraction buffer (Arcturus). RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus) and the samples were DNAse treated using RNase-Free DNase (Qiagen). Each slide capture session lasted no longer than 20 minutes to minimize RNA degradation. The laser capture settings were: 55 mW beam, 1.5 ms pulse, 15 µm spot size. Subsequently, RNA was amplified through two rounds of linear amplification using the MessageAmp aRNA Kit (Ambion, Inc). Sample quality and quantification was assessed by agarose gel electrophoresis and absorbance at A260.
|
| Label |
Cy5
|
| Label protocol |
The protocol used for indirect labeling of cDNAs was a modification of a protocol described elsewhere (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm). In brief, cDNA probes were made from 2 µg of amplified RNA in a reaction volume of 30 µl containing 5 µg random hexamer primers, 0.2 mM 5-(3-aminoallyl)-2_-deoxyuridine-5_-triphosphate (amino acid-dUTP; Sigma-Aldrich), 0.3 mM dTTP, 0.5 mM each dATP, dCTP, and dGTP, and 380 units of Superscript II reverse transcriptase (Invitrogen) incubated at 42oC for 120 minutes. After RNA hydrolysis, purified cDNA was combined with either Cy3 or Cy5 monoreactive fluors (Amersham) that covalently couple to the cDNA-incorporated aminoallyl linker in the presence of 50 mM NaHCO3 (pH 9.0). Samples were randomly labeled with either Cy3 or Cy5 dye to account for dye-bias. The coupling reaction was quenched with hydroxylamine.
|
| |
|
| Channel 2 |
| Source name |
Prostate_cancer_03-155C_LCM_GL4
|
| Organism |
Homo sapiens |
| Characteristics |
Prostate Cancer patient 03-155C Gleason_Score:4+4 (tertiary pattern 3)
LCM_Gleason_Pattern:4 Gleason_Pattern:4 Age:60-69 PSA:6.2 Volume:4.5 Margin_Status:negative Treatment:low-fat diet (study patient)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following LCM, captured cells were lysed in RNA extraction buffer (Arcturus). RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus) and the samples were DNAse treated using RNase-Free DNase (Qiagen). Each slide capture session lasted no longer than 20 minutes to minimize RNA degradation. The laser capture settings were: 55 mW beam, 1.5 ms pulse, 15 µm spot size. Subsequently, RNA was amplified through two rounds of linear amplification using the MessageAmp aRNA Kit (Ambion, Inc). Sample quality and quantification was assessed by agarose gel electrophoresis and absorbance at A260.
|
| Label |
Cy3
|
| Label protocol |
The protocol used for indirect labeling of cDNAs was a modification of a protocol described elsewhere (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm). In brief, cDNA probes were made from 2 µg of amplified RNA in a reaction volume of 30 µl containing 5 µg random hexamer primers, 0.2 mM 5-(3-aminoallyl)-2_-deoxyuridine-5_-triphosphate (amino acid-dUTP; Sigma-Aldrich), 0.3 mM dTTP, 0.5 mM each dATP, dCTP, and dGTP, and 380 units of Superscript II reverse transcriptase (Invitrogen) incubated at 42oC for 120 minutes. After RNA hydrolysis, purified cDNA was combined with either Cy3 or Cy5 monoreactive fluors (Amersham) that covalently couple to the cDNA-incorporated aminoallyl linker in the presence of 50 mM NaHCO3 (pH 9.0). Samples were randomly labeled with either Cy3 or Cy5 dye to account for dye-bias. The coupling reaction was quenched with hydroxylamine.
|
| |
|
| |
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 using a GenePix 4000B fluorescent scanner (Axon Instruments, Foster City, CA). The image intensity data were gridded and extracted using GenePix Pro 4.1 software.
|
| Description |
Human prostate cancer patient 03-155C with Gleason Score 4+4 (tertiary pattern 3) prostate cancer hybridized against normal adjacent prostate tissue from the same patient through laser capture microdissection (LCM).
|
| Data processing |
Log-Ratios: For each spot and in each channel (Cy3 and Cy5); the median background intensity was subtracted from the median foreground intensity. Log-ratios of cancer expression to benign expression were created by first dividing the background subtracted intensities (CaP/Benign) and then taking the log base 2. If the median background intensity was greater than the median foreground intensity, the spot was considered missing. Removal of Control Genes: The array contained approximately 400 clones used only for quality control purposes (i.e. yeast sequences or blank spots). These clones were removed from the dataset. Lowess Normalization: For each array, the log-ratio data were centered using a print-tip specific Lowess curve (Y. H. Yang, S. Dudoit, P. Luu and T. P. Speed. Normalization for cDNA Microarray Data. SPIE BiOS 2001, San Jose, California, January 2001). This curve was fit to the log intensity versus log-ratio plot using the neighboring 20.0% of the data to calculate the fit at each spot. The Lowess fit at each point was subtracted from the observed log-ratio for that spot, resulting in a normalized log-ratio. Assessing Spot Quality: Spots of poor quality, as determined by both visual inspection and GenePix Pro 4.1 quality flags were considered missing. In addition, spots with background subtracted intensity levels less than 300 were considered missing due to poorly hybridized cDNAs. Clones which were missing on > 20% of arrays were removed from the analysis. Imputation: Missing values were imputed using k-nearest neighbors imputation (k = 10) (2). The dataset was split by Gleason pattern (Grades 3, 4 and 5) and imputation was performed separately for each pattern. Average Replicated Clones: Log-ratios from the replicated cDNA spots on each PEDB chip were averaged after normalization and imputation. These average expression values were used for comparative analysis.
|
| |
|
| Submission date |
Jun 21, 2006 |
| Last update date |
Oct 02, 2008 |
| Contact name |
Denise Mauldin |
| E-mail(s) |
dmauldin@fhcrc.org
|
| Phone |
2066673480
|
| Fax |
2066672917
|
| URL |
http://www.pedb.org
|
| Organization name |
Fred Hutchinson Cancer Research Center
|
| Department |
Human Biology
|
| Lab |
Peter Nelson
|
| Street address |
1100 Fairview Ave N D4-100
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL3834 |
| Series (1) |
| GSE5132 |
Molecular Correlate to Gleason Grade in Prostate Adenocarcinoma |
|
| Data table header descriptions |
| ID_REF |
the unique identifier of the feature derived from the Array List |
| VALUE |
same as UNF_VALUE but with flagged values removed |
| X |
the X-coordinate in µm of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image. |
| Y |
the Y-coordinate in µm of the center of the feature-indicator associated with the feature, where (0,0) is the top left of the image. |
| Dia. |
the diameter in µm of the feature-indicator. |
| F635 Median |
median feature pixel intensity at wavelength #1 (635 nm). |
| F635 Mean |
mean feature pixel intensity at wavelength #1 (635 nm). |
| F635 SD |
the standard deviation of the feature pixel intensity at wavelength #1 (635 nm). |
| B635 Median |
the median feature background intensity at wavelength #1 (635 nm). |
| B635 Mean |
the mean feature background intensity at wavelength #1 (635 nm). |
| B635 SD |
the standard deviation of the feature background intensity at wavelength #1 (635 nm). |
| % > B635+1SD |
the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #1 (635 nm). |
| % > B635+2SD |
the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #1 (635 nm). |
| F635 % Sat. |
the percentage of feature pixels at wavelength 635 that are saturated. |
| F532 Median |
median feature pixel intensity at wavelength #2 (532 nm). |
| F532 Mean |
mean feature pixel intensity at wavelength #2 (532 nm). |
| F532 SD |
the standard deviation of the feature intensity at wavelength #2 (532 nm). |
| B532 Median |
the median feature background intensity at wavelength #2 (532 nm). |
| B532 Mean |
the mean feature background intensity at wavelength #2 (532 nm). |
| B532 SD |
the standard deviation of the feature background intensity at wavelength #2 (532 nm). |
| % > B532+1SD |
the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength #2 (532 nm). |
| % > B532+2SD |
the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength #2 (532 nm). |
| F532 % Sat. |
the percentage of feature pixels at wavelength 532 that are saturated. |
| Ratio of Medians (635/532) |
the ratio of the median intensities of each feature for each wavelength, with the median background subtracted. |
| Ratio of Means (635/532) |
the ratio of the arithmetic mean intensities of each feature for each wavelength, with the median background subtracted. |
| Median of Ratios (635/532) |
the median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted. |
| Mean of Ratios (635/532) |
the geometric mean of the pixel-by-pixel ratios of pixel intensities, with the median background subtracted. |
| Ratios SD (635/532) |
the geometric standard deviation of the pixel intensity ratios. |
| Rgn Ratio (635/532) |
the regression ratio of every pixel in a 2-feature-diameter circle around the center of the feature. |
| Rgn R² (635/532) |
the coefficient of determination for the current regression value. |
| F Pixels |
the total number of feature pixels. |
| B Pixels |
the total number of background pixels. |
| Sum of Medians |
the sum of the median intensities for each wavelength, with the median background subtracted. |
| Sum of Means |
the sum of the arithmetic mean intensities for each wavelength, with the median background subtracted. |
| Log Ratio (635/532) |
log (base 2) transform of the ratio of the medians. |
| F635 Median - B635 |
the median feature pixel intensity at 635 with the median background subtracted. |
| F532 Median - B532 |
the median feature pixel intensity at 532 with the median background subtracted. |
| F635 Mean - B635 |
the mean feature pixel intensity at 635 with the median background subtracted. |
| F532 Mean - B532 |
the mean feature pixel intensity at 532 with the median background subtracted. |
| F635 Total Intensity |
the sum of all pixel intensities in the feature at wavelenth 635. |
| F532 Total Intensity |
the sum of all pixel intensities in the feature at wavelenth 532. |
| SNR 635 |
the signal-to-noise ratio of the feature, calculated as (F635 Mean - B635 Mean)/B635 SD. |
| SNR 532 |
the signal-to-noise ratio of the feature, calculated as (F532 Mean - B532 Mean)/B532 SD. |
| Flags |
the type of flag associated with a feature; GenePix Pro 4.1 quality flags 0, -50, -75 |
| Normalize |
the normalization status of the feature (included/not included). |
| INV_VALUE |
|
| UNF_VALUE |
-[INV_VALUE]; Lowess-normalized, log2(CaP/Benign) ratios |
