|
Status |
Public on Dec 01, 2008 |
Title |
Human Prostate GSK-19 |
Sample type |
RNA |
|
|
Channel 1 |
Source name |
Reference
|
Organism |
Homo sapiens |
Characteristics |
Reference
|
Extracted molecule |
total RNA |
Extraction protocol |
Prostate samples embedded in OCT were used for laser capture microdissection (LCM). Approximately 2000-3000 epithelial cells per sample were collected from 8µm sections using the Arcturus Veritas™ Laser Capture Microdissection System according to the Arcturus HistoGene LCM Frozen Section Staining Kit Protocol (Mountain View, CA). Total RNA was isolated using the Arcturus Picopure™ Kit, and the samples were treated with DNAse using the Qiagen RNase-Free DNase Set (Qiagen Inc, Valencia, CA). Total RNA was subjected to two rounds of linear amplification using the Ambion MessageAmpII Kit (Ambion Inc, Austin, TX), quantitated in a Gene-Spec III spectrophotometer (Hitachi, Tokyo) and aRNA integrity evaluated using gel electrophoresis.
|
Label |
Cy5
|
Label protocol |
Complementary DNA (cDNA) probe pairs were prepared by amino-allyl reverse transcription using 2 µg of amplified RNA from microdissected samples and 30 µg of total RNA from a reference RNA pool composed of total RNA isolated from the LNCaP, DU145, and PC3 prostate cell lines. Probes were labeled with either Cy5 or Cy3 fluors (Amersham Bioscience, Piscataway, NJ) and competitively hybridized to custom cDNA microarrays spotted in duplicate with approximately 6,700 unique cDNA clones from the Prostate Expression Database (PEDB) as previously described in Pritchard CC, Hsu L, Delrow J, and Nelson PS. Project normal: defining normal variance in mouse gene expression. Proc Natl Acad Sci U S A 2001;98:13266-13271.
|
|
|
Channel 2 |
Source name |
Human Prostate GSK-19 treated with Immediate Radical Prostatectomy
|
Organism |
Homo sapiens |
Characteristics |
Human Prostate GSK-19. Specimen ID: GSK-19; Treatment: Immediate Radical Prostatectomy; Reference: HGSv5;
|
Extracted molecule |
total RNA |
Extraction protocol |
Prostate samples embedded in OCT were used for laser capture microdissection (LCM). Approximately 2000-3000 epithelial cells per sample were collected from 8µm sections using the Arcturus Veritas™ Laser Capture Microdissection System according to the Arcturus HistoGene LCM Frozen Section Staining Kit Protocol (Mountain View, CA). Total RNA was isolated using the Arcturus Picopure™ Kit, and the samples were treated with DNAse using the Qiagen RNase-Free DNase Set (Qiagen Inc, Valencia, CA). Total RNA was subjected to two rounds of linear amplification using the Ambion MessageAmpII Kit (Ambion Inc, Austin, TX), quantitated in a Gene-Spec III spectrophotometer (Hitachi, Tokyo) and aRNA integrity evaluated using gel electrophoresis.
|
Label |
Cy3
|
Label protocol |
Complementary DNA (cDNA) probe pairs were prepared by amino-allyl reverse transcription using 2 µg of amplified RNA from microdissected samples and 30 µg of total RNA from a reference RNA pool composed of total RNA isolated from the LNCaP, DU145, and PC3 prostate cell lines. Probes were labeled with either Cy5 or Cy3 fluors (Amersham Bioscience, Piscataway, NJ) and competitively hybridized to custom cDNA microarrays spotted in duplicate with approximately 6,700 unique cDNA clones from the Prostate Expression Database (PEDB) as previously described in Pritchard CC, Hsu L, Delrow J, and Nelson PS. Project normal: defining normal variance in mouse gene expression. Proc Natl Acad Sci U S A 2001;98:13266-13271.
|
|
|
|
Hybridization protocol |
Probes were labeled with either Cy5 or Cy3 fluors (Amersham Bioscience, Piscataway, NJ) and competitively hybridized to custom cDNA microarrays spotted in duplicate with approximately 6,700 unique cDNA clones from the Prostate Expression Database (PEDB) as previously described in Nelson PS, Pritchard C, Abbott D, and Clegg N. The human (PEDB) and mouse (mPEDB) Prostate Expression Databases. Nucleic Acids Res 2002;30:218-220 and Pritchard CC, Hsu L, Delrow J, and Nelson PS. Project normal: defining normal variance in mouse gene expression. Proc Natl Acad Sci U S A 2001;98:13266-13271.
|
Scan protocol |
Fluorescence array images were collected using a GenePix 4000B fluorescent scanner (Axon Instruments, Foster City, CA) and processed as we have previously described in True L, Coleman I, Hawley S, et al. A molecular correlate to the Gleason grading system for prostate adenocarcinoma. Proc Natl Acad Sci U S A 2006;103:10991-10996.
|
Description |
Human Prostate GSK-19 from Human hybridized against Reference
|
Data processing |
Changes in gene expression were evaluated using the Statistical Analysis of Microarray (SAM) program (http://www-stat.stanford.edu/_tibs/SAM/) to perform a one-sample t-test assessing the effect of Acyline treatment on gene expression (22). An FDR (false discovery rate) of less than 5% was considered significant. Quantile normalization was performed in Bioconductor (23) to allow single channel comparison of the absolute signal intensities in the experimental channel across different arrays in the same experiment.
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|
|
Submission date |
Dec 18, 2007 |
Last update date |
Dec 28, 2007 |
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 |
GPL4768 |
Series (1) |
GSE9972 |
Prostate Cancer Gene Expression Changes after Treatment with Neoadjuvant Dutasteride |
|
Data table header descriptions |
ID_REF |
the unique identifier of the feature derived from the Array List (up to 40 characters long, contained in quotation marks). |
VALUE |
normalized log (experiment/reference) ratio data comparable across rows and samples |
MetaCol |
Column referring to overall organization |
MetaRow |
Row referring to overall organization |
Column |
the column number of the feature. |
Row |
the row number of the feature. |
Name |
the name of the feature derived from the Array List (up to 40 characters long, contained in quotation marks). |
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. |
Normalize |
the normalization status of the feature (included/not included). |