|
| Status |
Public on Sep 18, 2012 |
| Title |
ULL-D-074 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Stratagene Reference, ampl. totRNA
|
| Organism |
Homo sapiens |
| Characteristics |
reference: Stratagene Reference, amplified total RNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
not provided
|
| Label |
Cy3
|
| Label protocol |
not provided
|
| |
|
| Channel 2 |
| Source name |
ULL-D-074, ampl. totRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary breast carcinoma
ID: ULL-D-074
|
| Growth protocol |
A Series of 212 primary breast cancer cases were studied; 80 of these tumors were analyzed using cDNA microarrays, along with one normal breast tissue sample collected from breast reduction surgery. Patient samples were sequentially collected at Ullev?l University Hospital from 1990 to 1994 (IRB approval 350, protocol 75026). The last update of patient information was done in 2006, providing an observation time of 12 to 16 years. Patients were followed until death or emigration, and only 12 patients were lost to follow-up. The average age of the 80 cases analyzed by cDNA microarrays was 65.0 years at time of primary surgery (range 28.2 to 87.7 years), similar to the average age of 64.4 years (range 28.2 to 91.5 years) for the total series. The 80 cases were selected from the total series based only on sufficient amount of fresh frozen tissue for microarray analysis. Consequently, a slightly higher fraction of patients with larger tumor size was observed in this subcohort. A summary of the clinical and histopathological data of the patients is shown in Table 1 (see Additional file 1 for more detailed information). All patients were treated according to Norwegian national guidelines at the time of diagnosis [19]. Patients receiving adjuvant systemic therapy were given nine courses of CMF (cyclophosphamide, methotrexate, 5-fluorouracil) and/or Tamoxifen for two years. Dosage of radiation given as adjuvant treatment was dependent on indication; after breast conserving therapy the mammary gland was given 50 Gy (2 Gy ? 25). The number of samples entered into the survival analyses is smaller than 212 (full dataset) and 80 (subset with gene expression data); excluding patients with missing information or distant metastases at the time of diagnosis and primary surgery, leaves us with a maximum number of 200 (full set) and 77 (subset) patients. <br> Primary breast carcinoma tissue was snap frozen and stored at -80?C. Frozen sections stained with hematoxylin/eosin were reviewed to confirm tumor content, and specimens in which at least 5% of the cells were carcinoma cells were included in this study. The majority of samples (80%) analyzed using microarrays had at least 40% tumor cell content.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from snap frozen tumor tissue using TRIzol? solution (Invitrogen?, Carlsbad, California, USA). The concentration of total RNA was determined using an HP 8453 spectrophotometer (Hewlett Packard) and the integrity of the RNA was assessed using a 2100 Bioanalyzer (Agilent, Santa Clara, California, USA).
|
| Label |
Cy5
|
| Label protocol |
Jeffrey Lab RNA Amplification Protocol First strand cDNA synthesis Mix the following contents in 0.2 ml PCR tube and spin briefly: N microl (3 mg) Total RNA 1 microl (0.5 mg/ml) Eberwine primer* 9-N microl RNase-free H2O * Eberwine primer = T7-oligo-dT(15) (5?-AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC TTT TTT TTT TTT TTT-3?) Incubate in PCR machine at 70?C for 3 min, cool on ice for 2 min. Spin briefly. Add the following contents: 4 microl 5X first strand buffer 2 microl 0.1M DTT 1 microl RNasin 2 microl 10mM dNTP mix 2 microl Superscript?II Mix contents, spin briefly, and incubate at 42?C for 1.5 hours. Second strand cDNA synthesis Add the following contents to the first strand synthesis reaction: RNase-free H2O 106 microl 10X Advantage? PCR buffer (Clontech) 15 microl 10mM dNTP mix 3 microl RNase H (2U/microl) 1 microl Advantage? Polymerase Mix (Clontech) 3 microl Incubate in PCR machine at 37?C for 5 min, 94?C for 2 min, 65?C for 1 min, 75?C for 30 min. Stop reaction by adding 7.5 microl of 1M NaOH with 2mM EDTA and incubate in PCR machine at 65?C for 10 min. ds cDNA cleanup If necessary, transfer to a larger tube. Add 150 microl phenol:chloroform:isoamyl alcohol 25:24:1 and mix by pipetting. Transfer to phase lock gel tube and spin at ~16,000g for 5min. Transfer aqueous layer to new tube. Add 1 microl acrylamide (0.1 mg/ml), 70 microl 7.5M NH4Ac, 1 ml 100% EtOH. Spin at ~16,000g for 20 min at room temperature (not at 4?C). Wash pellet with 500 microl 75% EtOH, spin at ~16,000g for 5 min. Air dry, and suspend the pellet in 16 microl RNase-free H2O. In vitro transcription (IVT) Mix at room temperature (the spermidine in the 10X transcription buffer can coprecipitate the template DNA if the reaction is assembled on ice): 16 microl cDNA 4 microl 10X reaction buffer (room temperature) 16 microl 75mM NTP mix (on ice) 4 microl T7 enzyme mix* *Ambion T7 MEGAscript? kit Incubate at 37?C for 5 hours (we have tried incubating for 2-6 hours and achieved the highest correlation between amplified and unamplified samples at 5 hours incubation). aRNA cleanup (using Qiagen RNeasy? mini kit) Add 10 microl b-mercaptoethanol to 1ml buffer RLT before use (stable for 1 month). Transfer IVT reaction into a 1.5 ml new tube, add 60 microl RNase-free H2O, add 350 microl buffer RLT, mix by pipetting. Add 250 microl 100% EtOH, mix by pipetting. Apply sample to RNeasy? column, spin at ~12,000g for 15 sec at room temperature. Transfer column to new collection tube. Add 500 microl buffer RPE, spin at ~12,000g for 15 sec. Discard flow through, add 500 microl buffer RPE, spin at ~12,000g for 2 min. Transfer column to new collection tube, add 30 microl RNase-free H2O to the membrane, spin at ~12,000g for 1 min to elute Measure aRNA yield at A260;
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| |
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| |
| Hybridization protocol |
1. Prepare the reaction for each aRNA sample. aRNA Rhx Primer(8?ug/?ul) ddH2O Total Cy5 X ul(3 ug) 1 ul 16-(X+1) 16 ul Cy3 Y ul(3 ug) 1 ul 16-(Y+1) 16 ul 2. Heat to 70?C for 10 min and cool on ice. 3. Prepare reaction mixture for each Cye dye. 5X First-Strand Buffer 6 ?l 50X dNTPs 0.7 ul Cye Dyes 3 ul 0.1 M DTT 3 ul Superscript II 1.7 ul Total Volume 14.4 ul 4. Add 14.4 ul of reaction mixture to the 16 ul reaction tube, mix well, and incubate at 42?C for 1 hour. 5. Add another 1 ul of SSII to each sample and incubate at 42?C for 1 hour. 6. Add 15 ul of 0.1 N NaOH (with 2mM EDTA) and incubate at 65?C for 8 min. 7. Neutralize by adding 15 ul of 0.1 HCl. 8. Add 450 ?l of TE to a Microcon YM-30 Microcon. Add 15 ul of COT1 (1 mg/ml) DNA. 9. Add Cy5 and Cy3 labeled probe to the same Microcon. 10. Wash 1: Spin column for 7 min at 14,000 x g. Wash 2. Remove flow through and add 500 ?l TE Spin for 7 min. Wash 3. Repeat Wash 2 and concentrate probe to less than or equal to 30-35 ul. 11. Invert the Microcon into a clean 1.5 ml tube and spin for 2 min. at 14,000 RPM to recover the probe. 12. Transfer 26.1 ul of probe to new PCR tube: Sample 26.1ul Poly (A) DNA 2 ul (10 ug/ul) Yeast tRNA 1 ul (10 ug/ul) 20X SSC 5.3 ul 10% SDS 0.6 ul Total hybridization volume 35 ul 13. Denature probe by heating for 2 min at 92?C and 42?C for 20-30 min. 14. Spin briefly and place 35 ul to on array. 15. Place a coverslip (24 X 60 mm size) on to array and add 20-30 ul of 3X SSC to each corner of the hybridization chamber. 16. Hybridize at 65?C for 14-18 hours. 17. Wash slide once with 2X SSC/ 0.05% SDS, for 2 min. 18. Transfer slide to 1X SSC for 2 min. 19. Wash slide in 50?C 0.2X SSC for 2 min, twice. 20. Spin slide in 600RPM for 5min.
|
| Scan protocol |
Scanner Model: GenePix 4000B
|
| Description |
Simple annotation: Breast, Primary tumor
Image: http://smd.stanford.edu/MicroArray/gifs/2003-08/43896.gif
|
| Data processing |
VALUE is Log (base 2) of the ratio of the median of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm)
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| |
|
| Submission date |
Sep 18, 2012 |
| Last update date |
Sep 18, 2012 |
| Organization |
Stanford Microarray Database (SMD) |
| E-mail(s) |
array@genome.stanford.edu
|
| Phone |
650-498-6012
|
| URL |
http://genome-www5.stanford.edu/
|
| Department |
Stanford University, School of Medicine
|
| Street address |
300 Pasteur Drive
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL8650 |
| Series (1) |
| GSE40954 |
TP53 mutation status and gene expression profiles are powerful prognostic markers of breast cancer |
|
| Data table header descriptions |
| ID_REF |
ID_REF |
| CH1I_MEAN |
Mean feature pixel intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale |
| CH2I_MEAN |
Mean feature pixel intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale |
| CH1B_MEDIAN |
The median feature background intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel; Background |
| CH2B_MEDIAN |
The median feature background intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background |
| CH1D_MEAN |
The mean feature pixel intensity at wavelength 532 nm with the median background subtracted.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel |
| CH2D_MEAN |
.The mean feature pixel intensity at wavelength 635 nm with the median background subtracted.; Type: integer; Scale: linear_scale; Channel: Cy5 channel |
| CH1I_MEDIAN |
Median feature pixel intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale |
| CH2I_MEDIAN |
Median feature pixel intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale |
| CH1B_MEAN |
The mean feature background intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale; Background |
| CH2B_MEAN |
The mean feature background intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Background |
| CH1D_MEDIAN |
The median feature pixel intensity at wavelength 532 nm with the median background subtracted.; Type: integer; Scale: linear_scale |
| CH2D_MEDIAN |
The median feature pixel intensity at wavelength 635 nm with the median background subtracted.; Type: integer; Scale: linear_scale |
| CH1_PER_SAT |
The percentage of feature pixels at wavelength 532 nm that are saturated.; Type: integer; Scale: linear_scale |
| CH2_PER_SAT |
The percentage of feature pixels at wavelength 635 nm that are saturated.; Type: integer; Scale: linear_scale |
| CH1I_SD |
The standard deviation of the feature intensity at wavelength 532 nm.; Type: integer; Scale: linear_scale; Channel: Cy3 Channel |
| CH2I_SD |
The standard deviation of the feature pixel intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Channel: Cy5 channel |
| CH1B_SD |
The standard deviation of the feature background intensity at wavelength 532 nm.; Type: float; Scale: linear_scale; Channel: Cy3 Channel; Background |
| CH2B_SD |
The standard deviation of the feature background intensity at wavelength 635 nm.; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background |
| PERGTBCH1I_1SD |
The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale |
| PERGTBCH2I_1SD |
The percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity, at wavelength 635 nm.; Type: integer; Scale: linear_scale |
| PERGTBCH1I_2SD |
The percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale |
| PERGTBCH2I_2SD |
The percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity, at wavelength 532 nm.; Type: integer; Scale: linear_scale |
| SUM_MEAN |
The sum of the arithmetic mean intensities for each wavelength, with the median background subtracted.; Type: integer; Scale: linear_scale |
| SUM_MEDIAN |
The sum of the median intensities for each wavelength, with the median background subtracted.; Type: integer; Scale: linear_scale |
| RAT1_MEAN |
Ratio of the arithmetic mean intensities of each spot for each wavelength, with the median background subtracted. Channel 1/Channel 2 ratio, (CH1I_MEAN - CH1B_MEDIAN)/(CH2I_MEAN - CH2B_MEDIAN) or Green/Red ratio.; Type: float; Scale: linear_scale |
| RAT2_MEAN |
The ratio of the arithmetic mean intensities of each feature for each wavelength, with the median background subtracted.; Type: float; Scale: linear_scale |
| RAT2_MEDIAN |
The ratio of the median intensities of each feature for each wavelength, with the median background subtracted.; Type: float; Scale: linear_scale |
| PIX_RAT2_MEAN |
The geometric mean of the pixel-by-pixel ratios of pixel intensities, with the median background subtracted.; Type: float; Scale: linear_scale |
| PIX_RAT2_MEDIAN |
The median of pixel-by-pixel ratios of pixel intensities, with the median background subtracted.; Type: float; Scale: linear_scale |
| RAT2_SD |
The geometric standard deviation of the pixel intensity ratios.; Type: float; Scale: linear_scale |
| TOT_SPIX |
The total number of feature pixels.; Type: integer; Scale: linear_scale |
| TOT_BPIX |
The total number of background pixels.; Type: integer; Scale: linear_scale |
| REGR |
The regression ratio of every pixel in a 2-feature-diameter circle around the center of the feature.; Type: float; Scale: linear_scale |
| CORR |
The correlation between channel1 (Cy3) & Channel 2 (Cy5) pixels within the spot, and is a useful quality control parameter. Generally, high values imply better fit & good spot quality.; Type: float; Scale: linear_scale |
| DIAMETER |
The diameter in um of the feature-indicator.; Type: integer; Scale: linear_scale |
| X_COORD |
X-coordinate of the center of the spot-indicator associated with the spot, where (0,0) is the top left of the image.; Type: integer; Scale: linear_scale |
| Y_COORD |
Y-coordinate of the center of the spot-indicator associated with the spot, where (0,0) is the top left of the image.; Type: integer; Scale: linear_scale |
| TOP |
Box top: int(((centerX - radius) - Xoffset) / pixelSize).; Type: integer; Scale: linear_scale |
| BOT |
Box bottom: int(((centerX + radius) - Xoffset) / pixelSize).; Type: integer; Scale: linear_scale |
| LEFT |
Box left: int(((centerY - radius) - yoffset) / pixelSize).; Type: integer; Scale: linear_scale |
| RIGHT |
Box right: int(((centerY + radius) - yoffset) / pixelSize); Type: integer; Scale: linear_scale |
| FLAG |
The type of flag associated with a feature: -100 = user-flagged null spot; -50 = software-flagged null spot; 0 = spot valid.; Type: integer; Scale: linear_scale |
| CH2IN_MEAN |
Normalized value of mean Channel 2 (usually 635 nm) intensity (CH2I_MEAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel |
| CH2BN_MEDIAN |
Normalized value of median Channel 2 (usually 635 nm) background (CH2B_MEDIAN/Normalization factor).; Type: integer; Scale: linear_scale; Channel: Cy5 channel; Background |
| CH2DN_MEAN |
Normalized value of mean Channel 2 (usually 635 nm) intensity with normalized background subtracted (CH2IN_MEAN - CH2BN_MEDIAN).; Type: integer; Scale: linear_scale; Channel: Cy5 channel |
| RAT2N_MEAN |
Type: float; Scale: linear_scale |
| CH2IN_MEDIAN |
Normalized value of median Channel 2 (usually 635 nm) intensity (CH2I_MEDIAN/Normalization factor).; Type: integer; Scale: linear_scale |
| CH2DN_MEDIAN |
Normalized value of median Channel 2 (usually 635 nm) intensity with normalized background subtracted (CH2IN_MEDIAN - CH2BN_MEDIAN).; Type: integer; Scale: linear_scale |
| RAT1N_MEAN |
Ratio of the means of Channel 1 (usually 532 nm) intensity to normalized Channel 2 (usually 635 nm) intensity with median background subtracted (CH1D_MEAN/CH2DN_MEAN). Channel 1/Channel 2 ratio normalized or Green/Red ratio normalized.; Type: float; Scale: linear_scale |
| RAT2N_MEDIAN |
Channel 2/Channel 1 ratio normalized, RAT2_MEDIAN/Normalization factor or Red/Green median ratio normalized.; Type: float; Scale: linear_scale |
| LOG_RAT2N_MEAN |
Log (base 2) of the ratio of the mean of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm) [log (base 2) (RAT2N_MEAN)].; Type: float; Scale: log_base_2 |
| VALUE |
Log (base 2) of the ratio of the median of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm) [log (base 2) (RAT2N_MEDIAN)].; Type: float; Scale: log_base_2 |
