title: X03410_Sty_8

tag: Primary malignant neoplasm of female breast; MULTIFOCAL INFILTRATING DUCT CARCINOMA, BREAST, ER- PR-; WITH PAGET'S DISEASE, AND METASTASIS TO REGIONAL NODES; ARISING IN A BACKGROUND OF FIBROCYSTIC DISEASE OF BREAST., 75-90


title: X05396_Sty_20

status: Public on Mar 17 2008

type: genomic

city: South San Francisco

first: Peter

last: Haverty

state: CA

address: 1 DNA Way

department: Bioinformatics

country: USA

institute: Genentech, Inc.

hyb_protocol: Mapping 500K arrays were hybridized according to the Affymetrix protocol (GeneChip Mapping 500K Assay Manual, Affymetrix, 2005) using an Affymetrix model 640 hybridization oven.

scan_protocol: Mapping 500K arrays were scanned according to the Affymetrix protocol (GeneChip Mapping 500K Assay Manual, Affymetrix, 2005) using a GS3000 scanner.

data_processing: Affymetrix 500K Mapping Array intensity signal CEL files were processed by dChip 2005 (Build date Nov 30, 2005) using the PM/MM difference model and invariant set normalization.  Forty-eight normal samples were downloaded from the Affymetrix website (http://www.affymetrix.com/support/technical/byproduct.affx?product=500k) and analyzed at the same time.  One CEL file for each set (Sty and Nsp) with the median signal intensity across the set was selected as the reference array.   The dChip-normalized signal intensities were converted to log2 ratios and segmented as follows.  For each autosomal probe set, the log2 tumor/normal ratio of each tumor sample was calculated using the average intensity for each probe set in the normal set.  For Chromosome X, the average of the 20 normal female samples was used.   Each probe set was mapped to the genome, NCBI assembly version 35, using annotation provided by the Affymetrix web site.  The log2 ratios were centered to a median of zero and segmented using the GLAD package for the R statistical environment.

label: biotin

molecule: genomic DNA

organism: Homo sapiens

label_protocol: Following restriction enzyme digestion, biotin labeled DNA was prepared according to the Affymetrix protocol from 500ng of starting DNA (GeneChip Mapping 500K Assay Manual, Affymetrix, 2005). The fluidics station model FS450 was used.

extract_protocol: A small piece of frozen tissue was removed from a sample of frozen tissue and incubated in a proteinase K digestion solution followed by cleanup using the Qiagen M48 robotics protocol.

tag: Primary malignant neoplasm of female breast; BREAST, LOCAL EXCISION

value: INFILTRATING DUCTAL CARCINOMA, TWO NODULES (1.8 AND 1.1 CM), BLOOM-RICHARDSON SCORE 3/3/3 = 9 (GRADE 3 OF 3).  ER- PR-., >90


title: X03221_Sty_2

tag: Primary malignant neoplasm of female breast; INFILTRATING DUCTAL CARCINOMA OF BREAST., 75-90


title: X04849_Sty_14

tag: Primary malignant neoplasm of female breast; BREAST

value: INFILTRATING DUCTAL CARCINOMA., >90


title: X41106_Nsp_47

source_name: breast-met

tag: Secondary malignant neoplasm of lung; LUNG, LUL LOBECTOMY

value: POORLY DIFFERENTIATED CARCINOMA C/W METASTASIS FROM PREVIOUS PRIMARY MEDULLARY CARCINOMA OF BREAST APPROXIMATELY NINE YEARS PRIOR.  NOTE: DONOR PREVIOUSLY TREATED WITH CHEMOTHERAPY., 75-90


title: X04860_Sty_16


title: X41110_Nsp_49

tag: Secondary malignant neoplasm of lymph node; SENTINEL AXILLARY LYMPH NODE

value: METASTATIC INFILTRATING DUCTAL CARCINOMA OF BREAST.  RECEPTOR STATUS OF PRIMARY TUMOR NOT STATED., 75-90


title: X03399_Sty_7


title: X03233_Sty_3

tag: Primary malignant neoplasm of female breast; INFILTRATING DUCTAL CARCINOMA OF BREAST., >90


title: X03195_Sty_1


title: X41103_Nsp_46

tag: Primary malignant neoplasm of female breast; MASTECTOMY

value: INFILTRATING DUCTAL CARCINOMA, 5 CM DIAMETER, METASTATIC TO 2 REGIONAL NODES.  PATIENT IS S/P MASTECTOMY AND RADIATION TREATMENT FOR INFILTRATING DUCTAL CARCINOMA OF OPPOSITE BREAST 13 YEARS PREVIOUSLY.  PRIOR TUMOR WAS ER+ PR+ (HER2/NEU NOT ASSESSED AT THAT TIME).  CURRENT TUMOR IS ER BORDERLINE +  PR+  HER2/NEU -.  TUMOR ARISES IN A BACKGROUND OF FIBROCYSTIC DISEASE OF BREAST., 75-90


title: X41108_Nsp_48

tag: Primary malignant neoplasm of female breast; PER SUBMITTED REPORT, MASTECTOMY

value: INFILTRATING CARCINOMA WITH DUCTAL AND LOBULAR FEATURES, 3.5 CM DIAMETER, METASTATIC TO ONE REGIONAL NODE.  ON GLGC PATHOLOGY REVIEW, FAVOR INTERPRETATION AS INFILTRATING DUCTAL CARCINOMA., 75-90


title: X41074_Nsp_44

tag: Primary malignant neoplasm of female breast; PARTIAL MASTECTOMY

value: INFILTRATING DUCTAL CARCINOMA, PAPILLARY TYPE, 3.5 CM DIAMETER.  INCOMPLETE INFORMATION FOR BLOOM-RICHARDSON GRADING, AND NO REPORT ON NODAL STATUS.  TUMOR ARISES IN FIBROCYSTIC BACKGROUND., 75-90


title: X59333_Nsp_51

tag: Primary malignant neoplasm of female breast; BREAST, LUMPECTOMY

value: INVASIVE DUCTAL CARCINOMA OF BREAST (2.5 CM), ACCOMPANIED BY LOBULAR CARCINOMA IN SITU.  METASTASES TO 4 OF 7 REGIONAL NODES IDENTIFIED.  NOTE: TUMOR STAGING IS ACCORDING TO THE 6TH EDITION OF THE AJCC CANCER STAGING MANUAL., 75-90


title: X20109_Nsp_39

tag: Secondary malignant neoplasm of brain; BRAIN (CEREBELLUM), BIOPSY

value: METASTATIC ADENOCARCINOMA, C/W BREAST PRIMARY (ALTHOUGH OTHER PRIMARY SITES NOT EXCLUDED)., 75-90


title: X20059_Nsp_38

value: INFILTRATING DUCT CARCINOMA OF BREAST (3.0 CM), METASTATIC TO 1 OF 23 LEVEL I NODES.  ALL 8 LEVEL II NODES NEGATIVE FOR TUMOR.  20% IN SITU COMPONENT PRESENT., 75-90


title: X41078_Nsp_45

tag: Primary malignant neoplasm of female breast; COMPLETION MASTECTOMY

value: RECURRENT INFILTRATING DUCTAL CARCINOMA, 7 YEARS AFTER PARTIAL MASTECTOMY FOR INITIAL PRIMARY TUMOR.  STAGING IS FOR INITIAL PRIMARY;  AT TIME OF RECURRENCE, ONE RESIDUAL AXILLARY NODE PRESENT, NEGATIVE FOR TUMOR.  INITIAL PRIMARY WAS ER+ PR+ HER2/NEU WEAK+; RECURRENCE (REPORTED IN CLINICAL DATA FOR THIS SAMPLE) IS ER- PR- HER2/NEU MODERATE+., 75-90


title: X14460_Nsp_36

value: INFILTRATING DUCTAL CARCINOMA, 8 CM DIAMETER, ULCERATING SKIN WITH EXTENSIVE LYMPHOVASCULAR INVASION, DISCRETE AXILLARY TUMOR NODULE PRESENT (? REPLACED LYMPH NODE); SYNCHRONOUS METASTASIS TO LUNG; ER- PR- HER2/NEU STRONG+., 75-90


title: X07287_Nsp_33

value: INFILTRATING DUCTAL CARCINOMA, 2 CM DIAMETER., 75-90


title: X07220_Nsp_30

value: INFILTRATING DUCTAL CARCINOMA, 4 CM DIAMETER., >90


title: X41073_Nsp_43

value: INFILTRATING DUCTAL CARCINOMA, MULTIFOCAL, BLOOM-RICHARDSON GRADE 3, WITH ULCERATION OF SKIN; METASTASIS TO AXILLARY NODES WITH EXTENSION INTO PERINODAL SOFT TISSUE., 75-90


title: X06002_Nsp_24

value: INFILTRATING DUCTAL CARCINOMA.  STAGE II., 75-90


title: X07226_Nsp_31

value: INFILTRATING DUCTAL CARCINOMA, 4.5 CM DIAMETER., 75-90


title: X37770_Nsp_41

tag: Primary malignant neoplasm of female breast; BREAST, EXCISIONAL BIOPSY

value: INFILTRATING DUCTAL CARCINOMA, 3 CM DIAMETER, NUCLEAR GRADE 2-3/3, HISTOLOGIC GRADE 2-3/3, MITOTIC RATE 3/3.  NO REPORT ON REGIONAL NODE STATUS., 75-90


title: X37760_Nsp_40

tag: Secondary malignant neoplasm of ovary; OVARY

value: METASTATIC CARCINOMA C/W METASTASIS FROM PRIMARY OF BREAST 28 MONTHS PREVIOUSLY.  PRIMARY WAS NUCLEAR GRADE 2, HISTOLOGIC GRADE 3, METASTATIC TO REGIONAL NODE AND LIVER AT INITIAL DIAGNOSIS; DONOR IS S/P HORMONAL THERAPY., 75-90


title: X07246_Nsp_32

tag: Primary malignant neoplasm of female breast; BREST

value: INFILTRATING DUCTAL CARCINOMA, 4 CM DIAMETER., 75-90


title: X05893_Nsp_21

value: INFILTRATING DUCTAL CARCINOMA.  STAGE II., >90


title: X07461_Nsp_34

value: INFILTRATING DUCTAL CARCINOMA AND DUCTAL CARCINOMA IN SITU., >90


title: X37777_Nsp_42

value: INFILTRATING DUCTAL CARCINOMA, 2.3 CM DIAMETER, NUCLEAR GRADE 3, HISTOLOGIC GRADE 3, METASTATIC TO 3 REGIONAL NODES., 75-90


title: X12894_Nsp_35

tag: Secondary malignant neoplasm of lymph node; AXILLARY NODE

value: METASTATIC INFILTRATING LOBULAR CARCINOMA OF BREAST.  DONOR PREVIOUSLY TREATED WITH CHEMOTHERAPY., >90


title: X05396_Nsp_20

value: INFILTRATING DUCTAL CARCINOMA, TWO NODULES (1.8 AND 1.1 CM), BLOOM-RICHARDSON SCORE 3/3/3 = 9 (GRADE 3 OF 3).  ER- PR-., >90


title: X04873_Nsp_18

value: INFILTRATING DUCTAL CARCINOMA., >90


title: X06196_Nsp_29

value: INFILTRATING DUCTAL CARCINOMA.  STAGE IV., >90


title: X06010_Nsp_28

value: INFILTRATING DUCTAL CARCINOMA.  STAGE IV., 75-90


title: X05895_Nsp_22

value: INFILTRATING DUCTAL CARCINOMA.  STAGE III., 75-90


title: X04877_Nsp_19

value: INFILTRATING DUCTAL CARCINOMA  WITH DOMINANT INTRADUCTAL COMPONENT., >90


title: X06008_Nsp_27

value: INFILTRATING DUCTAL CARCINOMA., 75-90


title: X06003_Nsp_25


title: X04866_Nsp_17


title: X06006_Nsp_26

value: INFILTRATING DUCTAL CARCINOMA.  STAGE I., >90


title: X04839_Nsp_13


title: X04837_Nsp_12

value: INFILTRATING DUCTAL CARCINOMA.., >90


title: X04834_Nsp_10


title: X04849_Nsp_14


title: X16683_Nsp_37

tag: Primary malignant neoplasm of female breast; MODIFIED RADICAL MASTECTOMY

value: IN SITU AND INFILTRATING DUCT CARCINOMA OF BREAST (4.1 CM).  REGIONAL NODES NEGATIVE FOR TUMOR., 75-90


title: X04835_Nsp_11

value: INFILTRATING APOCRINE CARCINOMA (SUBTYPE OF INFILTRATING DUCTAL CARCINOMA)., 75-90


title: X03399_Nsp_7


title: 9_V.F.M.3, Experimental replicate V

status: Public on Nov 09 2007

type: RNA

city: Blacksburg

first: sucheta

last: Tripathy

state: VA

address: 1, Washington street

department: Virginia  Bioinformatics Institute

institute: Virginia Tech

description: Data was collected from the hypocotyl regions of Soybean plants at different time points

hyb_protocol: hybridization cocktail is prepared, including the fragmented target, probe array controls, BSA, and herring sperm DNA. It is then hybridized to the probe array during a 16-hour incubation(Affymetrix technical manual - 2004)

scan_protocol: GeneChips were scanned using the Hewlett-Packard GeneChip Scanner 3000

data_processing: The data were analyzed with RMA suite from R package version 2.4.0. Background correction was using RMA followed by quantile normalization and data was summarized using median polish method

molecule: total RNA

organism:
Glycine max
Phytophthora sojae

source_name: Soybean cultivar PI291327 - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from total RNA. RNA is is first reverse transcribed using a T7-Oligo(dT) Promoter Primer in the first-strand cDNA synthesis reaction. Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA is purified and serves as a template in the subsequent in vitro transcription (IVT) reaction. The IVT reaction is carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling. The biotinylated cRNA targets are then cleaned up, fragmented, and hybridized to GeneChip expression arrays(Affymetrix Technical manual - 2004)

growth_protocol: Soybean plants were grown in growth chambers and later transferred to trays with clothes soaked in water

extract_protocol: QIAGEN RNeasy Kit : As recommended by manufacturer

treatment_protocol: Soybean plants were grown in growth chambers and transferred to trays prior to inoculation. There were 3 trays for each treatment type having 10 plants each. P.sojae materials were inoculated onto the roots of the host plant and RNA extraction was carried out either on 3rd day or 5th day post inoculation. For mock inoculation, the plants were inoculated with pure water with agar

tag: cultivar - PI291327

tag: Tissue/Cell Type

value: Soybean root/hypocotyls - P.sojae mycelium from minimal medium

tag: total Soybean RNA Plus 8 % P.sojae RNA added


title: X04832_Nsp_9

tag: Primary malignant neoplasm of female breast; BREAST


title: X05907_Nsp_23


title: X04860_Nsp_16


title: 9_Z.F.M.5, Experimental replicate Z

source_name: Soybean cultivar PI291327 - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation

tag: total Soybean RNA Plus 16 % P.sojae RNA added


title: X03235_Nsp_4

tag: Primary malignant neoplasm of female breast; INFILTRATING DUCTAL CARCINOMA OF BREAST., >90


title: X03233_Nsp_3


title: 9_W.K.I.3, Experimental replicate W

source_name: Soybean cultivar V710370 - pathogen(P.sojae) Inoculated: 72 hours post inoculation

tag: cultivar - V710370

tag: total Soybean RNA


title: X03239_Nsp_5


title: 5_Z.F.I.3, Experimental replicate Z

source_name: Soybean cultivar PI291327 - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 8_W.L.M.5, Experimental replicate W

source_name: Soybean cultivar WILLIAMS - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation

tag: cultivar - WILLIAMS


title: 7_Y.I.I.3, Experimental replicate Y

source_name: Soybean cultivar SLOAN - pathogen(P.sojae) Inoculated: 72 hours post inoculation

tag: cultivar - SLOAN


title: X03410_Nsp_8

tag: Primary malignant neoplasm of female breast; MULTIFOCAL INFILTRATING DUCT CARCINOMA, BREAST, ER- PR-; WITH PAGET'S DISEASE, AND METASTASIS TO REGIONAL NODES; ARISING IN A BACKGROUND OF FIBROCYSTIC DISEASE OF BREAST., 75-90


title: 9_Y.I.I.5, Experimental replicate Y

source_name: Soybean cultivar SLOAN - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 6_V.L.M.5, Experimental replicate V


title: 7_V.I.I.3, Experimental replicate V


title: 7_Z.K.I.5, Experimental replicate Z

source_name: Soybean cultivar V710370 - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: Pima S7 Cotton fiber Expression at 14dpa relative to 5dpa

status: Public on Jun 12 2008

city: Lubbock

first: Thea

middle: A

last: Wilkins

state: TX

address: 2500 Broadway,Campus Box 42122

department: Plant and Soil Sciences

institute: Texas Tech University

description: PDF files of detailed experimental protocols for RNA isolation (SOP MA002 v1.1), amino-allyl labeling (SOP MA003 v2.1) and microarray hybridization (SOP MA004 v2.1) can be accessed at Cotton Functional Genomics Center website.

hyb_protocol: The hybridization was performed according to Arpat et al., 2004. Printed slides were pre-hybridized in 5X SSC, 0.1% SDS, and 1 mg/µl BSA in a sealed coplin jar for 45 minutes at 42˚C, followed by two washes of 10 sec each in filtered ddH2O preheated to 42˚C, and dried with filtered compressed air. Alexa-labeled cDNA (500 ng/fluor or the equivalent of 40 to 45 pmol of dye depending upon the frequency of incorporation (FOI)) was suspended in 45µl of hybridization buffer (25% formamide, 4.16X SSC, 0.08% SDS, and 0.1 µg/µl sheared salmon sperm DNA) and loaded underneath the cover-slip (M Series LifterSlip, 22x60mm, Erie Scientific) using capillary action. Following hybridization at 42ºC for 16-18 hours in a humidified hybridization chamber (Genetix), slides were washed sequentially at 42ºC in 2X SSC, 0.2% SDS for 5 min, 0.5X SSC for 3 minutes, 4 times in 0.1X SSC for 1 minute per wash, and 0.05X SSC for 10 seconds, followed by drying with filtered compressed air.

scan_protocol: Hybridized slides were scanned using a GenPix3000 array scanner (Axon Instruments, CA, USA) followed by feature extraction to quantify signal intensities using ImaGene 4.2 software (BioDiscovery).

data_processing: Data were filtered and corrected for bias using the following steps: (1) Low quality spots (dust particles, scratches, high background, or irregularly shaped spots) were manually flagged and excluded. (2) Spot intensities were corrected by subtracting the mean of the background intensities from the signal intensity to reduce background noise. (3) Corrected spot intensities lower than the mean of corrected buffer spot plus 3 STDEV (Standard Deviation) were excluded (Hüser et al., 2003). (4) The absolute ratios (M) of dye swaps were correlated and the upper most 30% quartile of divergent data points were removed. This cut-off value enhanced the correlation between dye swaps by increasing the linearity. (5) Exclusion of all genes in which <60% of their data points did not pass all filtration steps. Reference calibration spots, which were spiked-in at a 1:1 ratio in both channels, were weighted to 10 before normalizing data. In order to adjust for effects that arise from variation inherent to microarray technology rather than biological differences in the RNA samples, filtered data was normalized using intensity-dependent normalization (Yang et al., 2002). The robust scatter plot smoother ‘Lowess’ implemented in R (version2.0.1) statistical software package and LIMMA (version 1.9.0) was used to perform a local per chip normalization. To make full use of within-array duplicated spots, LIMMA’s pooled correlation method (Symth et al., 2003) was used to estimate the strength of the correlation between duplicated spots by fitting separate linear models to the expression data for each gene, but with a common value for the between-replicate correlations. Normalized expression data at each time point was used as a reference point for all other time points (global reference) to calculate the relative expression, meaning that the expression at any given time point will be estimated independently six times relative to each of the other six time points.

label: Alexa Fluor® 555 and Alexa Fluor® 647 (Dye Swap included)

organism: Gossypium barbadense

source_name: Developing Cotton fiber at 14 days post anthesis (dpa)

label_protocol:
Total RNA samples were reverse transcribed and fluorescently labeled using the amino-allyl reverse transcription labeling protocol (Hughes et al., 2001; Arpat et al., 2004).
Total RNA (10 μg) spiked with 1000 pg of a reference mRNA spike mix (Lucidea Universal Scorecard, Amersham Pharmacia) was used to synthesize first-strand cDNA in the presence of amino allyl-modified dUTP (aa-dUTP) (Arpat et al., 2004, Hughes et al., 2001). The reverse transcription reaction mix contained ~10 µg “spiked” total RNA, 5g poly-T21VQ primers (Operon), 0.2 mM dNTPs (Sigma), 0.2 mM aminoallyl-dUTP/dUTP (Sigma) in a 2:3 ratio, and 2.5 U SuperScript III (Invitrogen). Following incubation for 2 hours at 45°C, the aminoallyl-labeled cDNA was purified using Microcon YM-30 columns (MilliPore) according to manufacturer’s instructions. Aminoallyl-cDNA was labeled using Alexa Fluor 555 and 647 succinimidyl ester dyes according to manufacturer’s instructions (Molecular Probes, Inc.), and purified using the QiaQuick PCR purification kit (Qiagen).

growth_protocol: G. barbadense L. cv. Pima S7, also known as Egyptian cotton, was grown in the green house under a 30/21°C day/night temperature regime in a randomized block design

extract_protocol: Total RNA was extracted using the modified hot borate method (Wan and Wilkins, 1994; Wilkins and Smart, 1996; Arpat et al., 2004)

tag: Flowers were tagged on the day of anthesis (0 days post anthesis (dpa)). Developing bolls from the first fruiting positions closest to the stem were collected at 14 dpa between 8 a.m. and 10 a.m. to eliminate diurnal effects. Bolls were collected from different plants, and immediately mingled, frozen in liquid Nitrogen, and then stored in 50-ml polypropylene tubes at -80°C.

source_name: Developing Cotton fiber at 5 days post anthesis (dpa)

tag: flowers were tagged on the day of anthesis (0 days post anthesis (dpa)). Developing bolls from the first fruiting positions closest to the stem were collected at 5 dpa between 8 a.m. and 10 a.m. to eliminate diurnal effects. Bolls were collected from different plants, and immediately mingled, frozen in liquid Nitrogen, and then stored in 50-ml polypropylene tubes at -80°C.


title: 6_Y.L.M.5, Experimental replicate Y


title: 6_Z.C.M.5, Experimental replicate Z

source_name: Soybean cultivar GENERAL - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation

tag: cultivar - GENERAL

tag: total Soybean RNA Plus 4 % P.sojae RNA added


title: 6_W.B.I.3, Experimental replicate W

source_name: Soybean cultivar CONRAD - pathogen(P.sojae) Inoculated: 72 hours post inoculation

tag: cultivar - CONRAD


title: 8_Z.E.M.3, Experimental replicate Z

source_name: Soybean cultivar OX20-8 - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation

tag: cultivar - OX20-8


title: 8_V.F.I.5, Experimental replicate V

source_name: Soybean cultivar PI291327 - pathogen(P.sojae) Inoculated: 120 hours post inoculation

tag: cultivar - PI291327


title: X04857_Nsp_15


title: X03344_Nsp_6

tag: Primary malignant neoplasm of female breast; INFILTRATING DUCTAL CARCINOMA OF BREAST.  , 75-90


title: X03195_Nsp_1


title: 5_Y.K.M.3, Experimental replicate Y

source_name: Soybean cultivar V710370 - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation

tag: Tissue/Cell Type

tag: total Soybean RNA Plus 2 % P.sojae RNA added


title: 5_W.C.M.3, Experimental replicate W

source_name: Soybean cultivar GENERAL - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 8_Y.E.I.5, Experimental replicate Y

source_name: Soybean cultivar OX20-8 - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 7_W.K.M.3, Experimental replicate W


title: X03221_Nsp_2

tag: Primary malignant neoplasm of female breast; INFILTRATING DUCTAL CARCINOMA OF BREAST., 75-90


title: 4_Z.B.I.5, Experimental replicate Z

source_name: Soybean cultivar CONRAD - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 5_V.C.M.3, Experimental replicate V


title: 4_W.E.I.5, Experimental replicate W


title: 3_W.I.I.3, Experimental replicate W


title: 32_Y.C.I.3, Experimental replicate Y

source_name: Soybean cultivar GENERAL - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 4_V.E.I.5, Experimental replicate V


title: 4_Y.A.M.5, Experimental replicate Y

source_name: Soybean cultivar ATHOW - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation

tag: cultivar - ATHOW


title: 32_V.L.M.3, Experimental replicate V

source_name: Soybean cultivar WILLIAMS - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation

tag: total Soybean RNA Plus 8 % P.sojae RNA added


title: 3_V.K.M.3, Experimental replicate V

tag: cultivar - V710370


title: 32_Z.B.I.3, Experimental replicate Z

tag: total Soybean RNA


title: 3_Z.A.I.3, Experimental replicate Z

source_name: Soybean cultivar ATHOW - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 3_Y.F.I.5, Experimental replicate Y


title: 31_W.L.I.3, Experimental replicate W

source_name: Soybean cultivar WILLIAMS - pathogen(P.sojae) Inoculated: 72 hours post inoculation

tag: cultivar - WILLIAMS


title: 31_Y.A.I.5, Experimental replicate Y

source_name: Soybean cultivar ATHOW - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 31_V.C.M.5, Experimental replicate V


title: 30_W.F.M.3, Experimental replicate W

source_name: Soybean cultivar PI291327 - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 30_V.F.I.3, Experimental replicate V

source_name: Soybean cultivar PI291327 - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 2_W.A.M.5, Experimental replicate W


title: 2_V.A.M.5, Experimental replicate V


title: 29_W.A.I.3, Experimental replicate W


title: 28_Z.E.M.5, Experimental replicate Z

source_name: Soybean cultivar OX20-8 - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation

tag: total Soybean RNA Plus 16 % P.sojae RNA added


title: 2_Y.B.I.3, Experimental replicate Y


title: 30_Y.B.M.3, Experimental replicate Y

source_name: Soybean cultivar CONRAD - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 29_Z.L.M.3, Experimental replicate Z


title: 29_Y.F.M.5, Experimental replicate Y

source_name: Soybean cultivar PI291327 - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 28_W.I.M.5, Experimental replicate W

source_name: Soybean cultivar SLOAN - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation

tag: cultivar - SLOAN


title: 27_Y.K.I.3, Experimental replicate Y

source_name: Soybean cultivar V710370 - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 27_V.B.I.5, Experimental replicate V


title: 2_Z.L.M.5, Experimental replicate Z

source_name: Soybean cultivar WILLIAMS - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 28_V.A.M.3, Experimental replicate V

source_name: Soybean cultivar ATHOW - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 27_Z.C.M.3, Experimental replicate Z

source_name: Soybean cultivar GENERAL - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 29_V.I.I.5, Experimental replicate V

source_name: Soybean cultivar SLOAN - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 26_V.E.I.3, Experimental replicate V

source_name: Soybean cultivar OX20-8 - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 27_W.E.M.3, Experimental replicate W

source_name: Soybean cultivar OX20-8 - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 30_Z.A.I.5, Experimental replicate Z


title: 26_Z.F.I.5, Experimental replicate Z

source_name: Soybean cultivar PI291327 - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 26_W.C.I.5, Experimental replicate W

source_name: Soybean cultivar GENERAL - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 28_Y.I.M.3, Experimental replicate Y

source_name: Soybean cultivar SLOAN - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 25_W.B.M.5, Experimental replicate W

source_name: Soybean cultivar CONRAD - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 26_Y.L.I.5, Experimental replicate Y

source_name: Soybean cultivar WILLIAMS - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 25_Z.K.I.3, Experimental replicate Z


title: 25_V.K.M.5, Experimental replicate V

source_name: Soybean cultivar V710370 - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 24_Y.I.M.5, Experimental replicate Y


title: 23_W.B.I.5, Experimental replicate W

source_name: Soybean cultivar CONRAD - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 24_W.F.I.3, Experimental replicate W

source_name: Soybean cultivar PI291327 - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 25_Y.E.M.5, Experimental replicate Y

source_name: Soybean cultivar OX20-8 - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 24_Z.C.I.5, Experimental replicate Z


title: 22_Y.K.I.5, Experimental replicate Y

source_name: Soybean cultivar V710370 - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 23_V.A.I.5, Experimental replicate V

source_name: Soybean cultivar ATHOW - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 21_W.I.I.5, Experimental replicate W


title: 23_Z.F.M.3, Experimental replicate Z

source_name: Soybean cultivar PI291327 - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 20_Y.C.I.5, Experimental replicate Y


title: 22_V.B.M.3, Experimental replicate V

source_name: Soybean cultivar CONRAD - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 20_W.A.M.3, Experimental replicate W

source_name: Soybean cultivar ATHOW - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 23_Y.E.M.3, Experimental replicate Y


title: 20_V.C.I.3, Experimental replicate V

source_name: Soybean cultivar GENERAL - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 22_Z.E.I.3, Experimental replicate Z

source_name: Soybean cultivar OX20-8 - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 21_Y.L.I.3, Experimental replicate Y

source_name: Soybean cultivar WILLIAMS - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 20_Z.A.M.3, Experimental replicate Z


title: 22_W.K.M.5, Experimental replicate W


title: 1_Y.C.M.3, Experimental replicate Y

source_name: Soybean cultivar GENERAL - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 31_Z.I.M.5, Experimental replicate Z

source_name: Soybean cultivar SLOAN - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 1_Z.I.M.3, Experimental replicate Z

source_name: Soybean cultivar SLOAN - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 21_Z.K.M.5, Experimental replicate Z


title: 32_W.K.I.5, Experimental replicate W


title: 21_V.E.M.5, Experimental replicate V


title: 1_V.B.I.3, Experimental replicate V

source_name: Soybean cultivar CONRAD - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 19_V.F.M.5, Experimental replicate V

source_name: Soybean cultivar PI291327 - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 1_W.F.I.5, Experimental replicate W

source_name: Soybean cultivar PI291327 - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 17_Z.L.I.5, Experimental replicate Z

source_name: Soybean cultivar WILLIAMS - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 16_Z.A.M.5, Experimental replicate Z

source_name: Soybean cultivar ATHOW - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 19_W.C.M.5, Experimental replicate W

source_name: Soybean cultivar GENERAL - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 19_Y.B.M.5, Experimental replicate Y

source_name: Soybean cultivar CONRAD - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 17_V.L.I.5, Experimental replicate V


title: 18_Z.I.I.3, Experimental replicate Z

source_name: Soybean cultivar SLOAN - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 19_Z.B.M.5, Experimental replicate Z


title: 16_W.B.M.3, Experimental replicate W


title: 18_V.I.M.3, Experimental replicate V


title: 17_Y.A.I.3, Experimental replicate Y

source_name: Soybean cultivar ATHOW - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 18_W.E.I.3, Experimental replicate W


title: 16_Y.A.M.3, Experimental replicate Y


title: 16_V.B.M.5, Experimental replicate V


title: 14_Y.F.I.3, Experimental replicate Y

source_name: Soybean cultivar PI291327 - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 18_Y.F.M.3, Experimental replicate Y

source_name: Soybean cultivar PI291327 - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 15_W.F.M.5, Experimental replicate W


title: 14_V.K.I.5, Experimental replicate V

source_name: Soybean cultivar V710370 - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 15_Y.C.M.5, Experimental replicate Y


title: 17_W.L.M.3, Experimental replicate W

source_name: Soybean cultivar WILLIAMS - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 15_V.E.M.3, Experimental replicate V

source_name: Soybean cultivar OX20-8 - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 14_Z.B.M.3, Experimental replicate Z

source_name: Soybean cultivar CONRAD - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation


title: 24_V.K.I.3, Experimental replicate V

source_name: Soybean cultivar V710370 - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 15_Z.L.I.3, Experimental replicate Z

source_name: Soybean cultivar WILLIAMS - pathogen(P.sojae) Inoculated: 72 hours post inoculation


title: 13_Z.I.I.5, Experimental replicate Z

source_name: Soybean cultivar SLOAN - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 14_W.L.I.5, Experimental replicate W


title: 13_V.A.I.3, Experimental replicate V


title: 289_LI_2

status: Public on May 31 2007

city: Amsterdam

first: Oscar

middle: Leonard

last: Volger

state: Noor Holland

address: Meibergdreef 15

department: Cardiology

country: Netherlands

institute: Academic Medical Center

description: See the methods section of the linked publication for additional information.

hyb_protocol: Each cRNA sample (1 micrograms; Cy5-labeled) was hybridized in duplicate against a common reference cRNA sample (1 micrograms; Cy3-labeled), for 16 hours at 40 degrees Celsius.

scan_protocol: Agilent scanner: resolution: 10 micrometer, scan area: 60 x 21.6 mm, PMT: 50%, Laser power: 50%

data_processing: After print-tip Loess-normalization (Loess span: 0.3; limma package, Bioconductor, available online: http://www.bioconductor.org), and background subtraction, the microarray intensity data imported in the Rosetta Resolver database.

molecule: polyA RNA

source_name: common reference

label_protocol: Within these cRNAs the molar ratio of incorporated aminoallyl-rUTP (A5660, Sigma, St. Louis, MO) to rUTP was 1:1. Cy3 mono-reactive dyes (PA23001, Amersham Biosciences, Piscataway, NJ) were coupled according to the manufacturer’s instructions. Labeled cRNA was purified using the RNeasy purification kit (Qiagen GmbH, Hilden, Germany).

extract_protocol: Total RNA of each one of the three reference components was isolated by Trizol, and treated with DNAse-I. Thereafter, mRNA of each component was linearly amplified for one round (MessageAmp aRNA kit, #1750, Ambion), synthesizing antisense cRNAs.

tag: The common reference cRNA sample comprised equal quantities of the following three components

value: (1) human umbilical cord endothelial cells (HUVEC), stimulated with TNF-alpha (50 microgram/ml, 6 hrs); (2) THP-1 cell line stimulated for 24 hours with phorbyl myristate (PMA, 100 microgram /ml), and subsequently for 6 hours with lipopolysaccharide (LPS, 1 microgram /ml), and (3) a mix of whole mount human aorta and iliac artery.

label_protocol: Within these cRNAs the molar ratio of incorporated aminoallyl-rUTP (A5660, Sigma, St. Louis, MO) to rUTP was 1:1. Cy5 mono-reactive dyes (PA25001, Amersham Biosciences, Piscataway, NJ) were coupled according to the manufacturer’s instructions. Labeled cRNA was purified using the RNeasy purification kit (Qiagen GmbH, Hilden, Germany).

extract_protocol: mRNA molecules from liver macrophages were linearly amplified (MessageAmp aRNA kit, #1750, Ambion), synthesizing antisense cRNAs.

tag: Liver macrophages


title: 289_LI_1

tag: Liver Macrophages


title: 13_Y.B.I.5, Experimental replicate Y

source_name: Soybean cultivar CONRAD - pathogen(P.sojae) Inoculated: 120 hours post inoculation

tag: cultivar - CONRAD


title: Newborn Mouse Ovary_Wild Type_1

status: Public on Oct 31 2007

city: Houston

first: Aleksandar

last: Rajkovic

address: One Baylor Plaza

institute: Baylor College of Medicine

description: Wild type mouse

hyb_protocol: HYBRIDIZATION COCKTAIL  Euk 20X must be heated 5 minutes before using it.  Vortex before using and spin down.  Put on ice when you’re finished with it but not before adding it to MasterMix. (multiply all by 3.5)	MasterMix components  2X Hybe Buffer			150.0 µL				525.0 µL DEPC H20			54.0 µL (to bring volume up to 300.0µL)	189.0 µL 100% DMSO			30.0 µL					105.0 µL Eukaryotic 20X Control	***	15.0 µL					52.5 µL Oligo B2			5.0 µL					17.5 µL Herring Sperm DNA		3.0 µL					10.5 µL BSA				3.0 µL					10.5 µL cRNAfragmented			40.0 µL (if you retrieve all from NanoDrop) you have these separate  1. Keep cRNA in tubes labeled FRAG. 2. Prepare MasterMix in 15mL corning tube. 3. Mix after each component by pipetting several times. 4. Add 260.0 µL of the Hybe MasterMix to each fragmented sample.  You total volume should be 300.0 µL. 5. If you are not hybridizing right away, store the cRNA-HybeCocktail mix in the -20°C until you’re ready to use it.  HYBRIDIZATION  Thaw Hybe Cocktails if they are frozen before heating. Pull arrays out of 4°C Deli Fridge 30 minutes before you want to load them.  They must be at room temperature before you pierce the septa to prevent leakage.  1. Put tube hats on to prevent popping open at high heat. 2. Place the tubes into the 95°C heating block for 5 minutes. 3. Move the tubes to the 45°C heating block, again for 5 minutes. 4. Spin in MicroFuge at max. speed for 5 minutes. 5. Load 250.0 µL into each array. 6. Put in Hybe Oven, set at 60rpm and 45°C for 16 hours.  After 16 hours of hybridization, remove the hybridization cocktail from the probe array and fill the probe array completely with the appropriate volume of Non-Stringent Wash Buffer (Wash Buffer A)  Wash Buffer A: Non-Stringent Wash Buffer  (6X SSPE, 0.01% Tween-20) For 1,000 mL: 300 mL of 20X SSPE 1.0 mL of 10% Tween-20 699 mL of water Filter through a 0.2 μm filter EukGE-WS2v5* Midi_euk2* 10 cycles of 2 mixes/cycle with Wash Buffer A at 30°C 6 cycles of 15 mixes/cycle with Wash Buffer B at 50°C Stain the probe array for 5 minutes in SAPE solution at 35°C 10 cycles of 4 mixes/cycle with Wash Buffer A at 30°C Stain the probe array for 5 minutes in antibody solution at 35°C Stain the probe array for 5 minutes in SAPE solution at 35°C 15 cycles of 4 mixes/cycle with Wash Buffer A at 35°C. The holding temperature is 25°C

scan_protocol: [Scanner] Pixel Size	1.56 hardware: GeneChip Scanner 3000 Affymetrix® Microarray Suite Filter	570 Pixel Size	1.56 Scanner ID	50101490 Number of Scans	1 Scanner Type	M10 As affymetrix protocol.

data_processing: Data in the value column is Mas5 scaled according to the algorithms of the genechip scanner, but is otherwise not modified, normalized, or processed.

label: Biotin

organism: Mus musculus

source_name: Postnatal Day 0.5 Ovarian Tissue WT

label_protocol:
Test RNA sample on NanoDrop.  Concentration must be 500_g/µL or more. Calculate the volume of RNA sample necessary to start protocol with 5µg. i.e. (5 µg) / (NanoDrop concentration reading in µg/µL) =      µL

RNA		  	 x uL
DEPC H20	      10-x uL
oligo T7	 	 2.0 µL

1. Add all components together in a 0.2µL strip tube.
2. Mix well and spin down.
3. Place in thermalcycler at 70°C for 10 minutes. Should be warming up to 70°C while you prep. the samples.
4. Allow thermalcycler to ramp down to 4°C.
5. 4°C for 2 minutes.  Spin down and put on ice.
6. Prepare the 1st Strand MasterMix.

Reverse Transcription (RT) 1st Strand

You should have thawed and vortexed the components during the 70°C-10 minute step.
5X 1st Strand Mix	(4.0 µL) /sample
0.1M DTT		(2.0 µL) /sample
10mM dNTPs		(1.0 µL) /sample
7.0 µL total
1. Add 7.0 µL 1st Strand MasterMix to each sample.  Mix.
2. Put on thermalcycler 42°C-1 Hour. Set timer for 2 minutes.
(This setting is actually programmed for 42°C for 1 hour and 3 minutes, to compensate for the extra 2
minutes at the beginning before adding the SuperScript II.
3. After 2 minutes at 42°C add 1.0 µL of SSII enzyme. (Addition should be done on thermalcycler)
4. The rest of the program will run, 1 hour at 42°C.
5. Proceed to 2nd Strand.
(components should be thawed ~10 minutes before thermalcycler finishes.)

Reverse Transcription (RT) 2nd Strand

DEPC H20				91.0 µL /sample
5X 2nd Strand Mix			30.0 µL /sample
E. coli DNA Polymerase I (DNA Pol I)	  4.0 µL /sample
10mM dNTPs				  3.0 µL /sample
E. coli DNA ligase (ligase)		  1.0 µL /sample
RNase H				  1.0 µL /sample

1. Put samples on thermalcycler, 16°C for 2 hours.
2. At 2 hours, add 2.0 µL of T4 DNA Polymerase.  Mix.
3. Run additional 5 minutes at 16°C.
4. Transfer samples to 1.5mL tube with 10.0 µL of 0.5M EDTA.  Vortex.
5. Use cDNA Clean-Up Module to clean up samples.

Clean-Up of Double Stranded cDNA

Necessary components:
1.5mL tube with cDNA and EDTA, mixed.
cDNA Binding Buffer
cDNA Wash Buffer (ethanol added by MCF)
cDNA Elution Buffer
cDNA clean-up column (in 4°C Deli Fridge)
Empty Collection Tube (provided in clean-up module)
Empty 1.5mL tube labeled with sample name for collecting final eluate.
MicroFuge 18 Centrifuge to spin down samples.

cDNA Wash Buffer is supplied as a concentrate and you have to add ethanol before using.  Check the tube before use to make sure that etOH has been added.

1. Label Collection Tube with sample name (on side).
2. Add 600.0 µL of cDNA Binding Buffer (yellow) into tube with sample and EDTA.
3. Vortex.  Spin down briefly (2 sec.)
4. Add 500.0 µL sample-Binding Buffer mix onto column.
5. Spin in MicroFuge 18 Centrifuge, 14,000rpm for 1 minute.
6. Pour eluate down sink.  Keep same collection tube.
7. Add the remaining cDNA-Binding Buffer mix onto column.
8. Spin 14K rpm for 1 minute.
9. Discard that collection tube and place column into new, labeled tube.
10. Add 720.0 µL cDNA Wash Buffer (ETOH added) to the column.
11. Spin 14K rpm for 1 minute.
12. Discard eluate into sink.  Replace column into collection tube.
13. Spin empty column with the lid open for 5 minutes at 14K rpm.
14. Place column into labeled 1.5mL tube.  Discard collection tube.
15. Air dry column for an additional minute (cover with KimWipe).
16. Add 14.0 µL Elution Buffer onto the center of the column.
17. Let sit at room temp for 1 minute.
18. Spin at 14K for 1 minute.
19. Discard column.
20. Measure the eluate volume.
21. Bring volume up to 20.0 µL with DEPC H20.
22. Store at -20ºC until ready to proceed with IVT step.

InVitro Transcription Reaction (IVT): production of biotin-labeled cRNA

cDNA should be at 20.0 µL before adding IVT MasterMix components.
IVT kit components should be thawed to room temp. ~20 minutes before using.
10X IVT Labeling Buffer (may have precipitate, mix well.) 	(4.0 µL) /sample
IVT Labeling NTP Mix					(12.0 µL) /sample
IVT Labeling Enzyme Mix	 			(4.0 µL) /sample

1. Mix components.  Spin briefly.
2. Add 20.0 µL IVT MasterMix to each sample.  Mix by pipetting.
3. 37 ºC for 16 hours in thermalcycler.
4. Clean-up cRNA with cRNA Clean-Up Module.

Clean-Up of Biotinylated cRNA with cRNA Clean-Up Module

Necessary Components:
cRNA Binding Buffer
cRNA Wash Buffer (ethanol added by MCF)
cRNA clean-up column (stored at 4°C in deli fridge)
Collection Tube (labeled with sample name on side)
DEPC H20
100% Ethanol
80% Ethanol
2 clean, labeled 1.5mL tubes

1. Transfer cRNA samples into clean, labeled 1.5mL tubes for clean-up.
2. Add 60.0 µL to each sample.  Vortex to mix and spin down.
3. Add 350.0 µL cRNA Binding Buffer to each sample.  Vortex and spin down.
4. Add 250.0 µL 100% ETOH to each sample.  Mix by pipetting.
5. Add cRNA-Binding Buffer-ETOH mix onto column (it all fits).
6. Spin at 14,000rpm on “Short Spin” setting (30 seconds).
7. Place columns into clean, labeled collection tube.  Discard previous tubes with eluate.
8. Add 500.0 µL cRNA Wash Buffer to each column.
9. Spin at 14K for “Short Spin”.
10. Discard eluate in sink but replace column into collection tube.
11. Add 500.0 µL 80% ETOH onto column.
12. “Short Spin” at 14K.
13. Discard eluate and replace columns into collection tube again.
14. Spin 14K for 5 minutes to dry column.
15. Put column into clean, labeled 1.5mL tube.  (This tube should have the request number, the term “IVT” and the sample name on top.  On the side put the PI last name and the date.)
16. Pipet 11.0 µL DEPC H20 onto center of column.
17. Spin 14K for 1 minute.
18. Add another 10.0 µL DEPC H20 onto center of column.
19. Spin 14K for 1 minute.
20. Measure volume of eluate.  Record the volume in your lab notebook.
21. NanoDrop samples.  Dilute with DEPC H20 if you need to, to get concentration into read-able range for NanoDrop (under 3000_g).
22. Make a dilution of the IVT samples for testing on the Agilent.

Perform calculations to determine adjusted cRNA amount.  Enter the total µg into MAID. Perform calculations for Fragmentation step and record the volumes in your lab notebook.  Calculations are done with the IVT NanoDrop adjusted cRNA concentrations acquired with the formula provided by Affy.

Fragmentation:

cRNA			      (X uL) 15ug
DEPC H20(variable)	 (32-x) µL
5X Fragmentation Buffer	   8.0  µL
40.0  uL
1. Add 8.0 µL 5X Frag. Buffer to each empty 1.5mL tube.
2. Add the required amount of water to each tube.  This amount will depend on the concentration of cRNA- how much cRNA to make 15 µg of fragmented cRNA, then how much DEPC H20 to bring the final volume to 40.0 µL?
3. Add the required amount of cRNA to each tube.
4. Make sure the 1.5mL tube is clearly labeled FRAG so that you can tell it apart from the IVT cRNA tube.
5. Put the FRAG tube onto the 99°C heat block for 35 minutes.
6. After fragmentation put the tube on ice.  Let it cool about 5 minutes on ice.
7. Spin in the MicroFuge at max. speed to get the condensation out of the lid.
8. Vortex and spin.
9. Test FRAG on NanoDrop.  Needs to be 15 µg or more.  (15 µg/40 µL=0.375 µg/µL or more)

Put the FRAG into hybridization cocktail and store at -20°C until you are ready to load it onto a chip.

extract_protocol: 50 ovaries were pooled per affymetrix array.  RNA was isolated using Qiagen RNeasy mini kits.  On column DNase digestion was used.  RNA was eluted in RNase free water and frozen at -80 degrees Celsius until sample quality could be assessed with the Agilent Bioanalyzer system.

treatment_protocol: Ovaries were dissected from day 0.5 animals and placed in RNAlater (Ambion) for 24 hours at 4 degrees Celsius.  After 24 hours, the RNAlater was removed and the ovaries were frozen on dry ice and placed in a -80 degree freezer.

tag: Background Srain

value: C57/BL6/129SVEV Hybrid

tag: Gender

value: Female

tag: Tissue

value: Ovary

tag: Age

value: .5 Days Postnatal


title: 12_Z.C.I.3, Experimental replicate Z

source_name: Soybean cultivar GENERAL - pathogen(P.sojae) Inoculated: 72 hours post inoculation

tag: cultivar - GENERAL

tag: Tissue/Cell Type


title: 289_SP_2

extract_protocol: mRNA molecules from splenic macrophages were linearly amplified (MessageAmp aRNA kit, #1750, Ambion), synthesizing antisense cRNAs.

tag: Splenic macrophages


title: 11_Y.L.M.3, Experimental replicate Y

tag: total Soybean RNA Plus 8 % P.sojae RNA added


title: 12_V.I.M.5, Experimental replicate V

source_name: Soybean cultivar SLOAN - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation


title: 12_Y.E.I.3, Experimental replicate Y

source_name: Soybean cultivar OX20-8 - pathogen(P.sojae) Inoculated: 72 hours post inoculation

tag: cultivar - OX20-8


title: Newborn Mouse Ovary_Nobox Null_1

description: Nobox is a homeobox gene expressed in oocytes

source_name: Postnatal Day 0.5 Ovarian Tissue Nobox Null


title: 11_Z.E.I.5, Experimental replicate Z

source_name: Soybean cultivar OX20-8 - pathogen(P.sojae) Inoculated: 120 hours post inoculation

tag: total Soybean RNA


title: 289_SP_1


title: 11_W.A.I.5, Experimental replicate W

source_name: Soybean cultivar ATHOW - pathogen(P.sojae) Inoculated: 120 hours post inoculation

tag: cultivar - ATHOW


title: 10_Z.K.M.3, Experimental replicate Z

source_name: Soybean cultivar V710370 - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation

tag: cultivar - V710370

tag: total Soybean RNA Plus 2 % P.sojae RNA added


title: 10_Y.K.M.5, Experimental replicate Y

source_name: Soybean cultivar V710370 - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation

tag: total Soybean RNA Plus 4 % P.sojae RNA added


title: 11_V.L.I.3, Experimental replicate V

tag: cultivar - WILLIAMS


title: 10_W.I.M.3, Experimental replicate W

source_name: Soybean cultivar SLOAN - Mock Inoculated (Inoculated with water + agar): 72 hours post inoculation

tag: cultivar - SLOAN


title: 12_W.C.I.3, Experimental replicate W


title: 10_V.C.I.5, Experimental replicate V

source_name: Soybean cultivar GENERAL - pathogen(P.sojae) Inoculated: 120 hours post inoculation


title: 288_LI_2

tag: The common reference cRNA sample comprised equal quantities of the following three components

tag: Liver macrophages


title: 289_CA_1

extract_protocol: Macrophages were isolated from human carotid artery plaque by Laser Capture Microdissection (Arcturus LCM system). After isolation of total RNA molecules, the samples were linearly amplified (MessageAmp aRNA kit, #1750, Ambion), synthesizing antisense cRNAs.


title: 289_CA_2


title: 288_SP_1


title: 288_SP_2


title: 288_LU_1

extract_protocol: Macrophages were isolated from Broncheo Alveolar Lavage (BAL) fluid. After isolation of total RNA molecules, the samples were linearly amplified (MessageAmp aRNA kit, #1750, Ambion), synthesizing antisense cRNAs.

tag: Lung macrophages


title: 288_LI_1


title: 288_CA_2


title: 288_LU_2

tag: Lung Macrophages


title: 289_LU_2

hyb_protocol: each cRNA sample (1 micrograms; Cy5-labeled) was hybridized in duplicate against a common reference cRNA sample (1 micrograms; Cy3-labeled), for 16 hours at 40 degrees Celsius.

data_processing: After print-tip Loess-normalization (Loess-span: 0.3; limma package, Bioconductor, available online: http://www.bioconductor.org), and background subtraction, the microarray intensity data were imported in the Rosetta Resolver database

value: (1) human umbilical cord endothelial cells (HUVEC), stimulated with TNF-alpha (50 microgram/ml, 6 hrs); (2) THP-1 cell line stimulated for 24 hours with phorbyl myristate (PMA, 100 microgram/ml), and subsequently for 6 hours with lipopolysaccharide (LPS, 1 microgram/ml), and (3) a mix of whole mount human aorta and iliac artery.

extract_protocol: Macrophages were isolated from Broncheo Alveolar Lavage (BAL) fluid. After isolation of total RNA molecules, the samples were linearly amplified(MessageAmp aRNA kit, #1750, Ambion), synthesizing antisense cRNAs.


title: CHC562N-U133A

status: Public on May 01 2007

city: Paris

first: Sandrine

last: Imbeaud

address: 15 rue de l'Ecole de Médecine

department: INSERM UMRS1138-EQUIPE 28, Sorbonne Université-Inserm-Université de Paris

country: France

institute: Centre de Recherche des Cordeliers

description: Detailed procedure is provided in the supplemental experimental procedures in [Rebouissou et al., J Biol Chem 2007, PMID: 17379603].

hyb_protocol: Hybridization was performed using 20 µg of cRNA per hybridization (GeneChip Fluidics Station 400), following the manufacturer’s protocols (Affymetrix, Santa Clara, CA).

scan_protocol: Array images were digitized by using Microarray Suite 5.0 (MAS5) software, embedded in the Affymetrix GeneChip Operating Software (Affymetrix, Santa Clara, USA).

data_processing: Gene intensity derivation was carried out from the raw numerical data (CEL files) by using the R package affy [Gautier et al., Bioinformatics 2004, 20: 307-15], available as part of the Bioconductor project [Gentleman et al., Genome Biol 2004, 5: R80], and the DNA-Chip analyzer (dChip) program [Li & Wong, PNAS 2001, 98: 31-6]. Probe sets corresponding to control genes or having a “_x_” annotation were masked yielding a total of 19,787 probe sets available for further analyses. Background subtraction, normalization and expression summaries for every probe set were calculated with the log-scale robust multi-array analysis (RMA) algorithm using a background adjusted PM intensities model [Irizarry et al., Nucleic Acids Research 2003, 31: e15], and with the model-based expression indexes (MBEI) calculation following a PM/MM difference model [Li & Wong, PNAS 2001, 98: 31-6].

label_protocol: For each case, 5 µg of total RNA were labeled according to the protocols provided by the manufacturer (Affymetrix, Santa Clara, CA).

extract_protocol: Frozen tissue sample specimens, in pieces from 50 to 150 mg each, were removed from the freezer, immediately placed into 700 µl of lysis Buffer RLT (Qiagen, Germany) together with stainless steel beads and homogenized using a Mixer Mill MM 300 (Qiagen, Germany), at 30 Hz over a period of 3 min. Then, homogenization was carried on as described in the commonly used RNeasy™ procedure.

tag: Sample

tag: Gender

value: female;

tag: Age at onset

value: 33 years;

tag: Disease

value: hepatocellular adenoma;

tag: Tissue

value: non tumoral liver.


title: CHC573N-U133A

value: 28 years;


title: CHC535N-U133A

value: 30 years;


title: daf2daf16_RepA

status: Public on Feb 08 2007

city: La Jolla

first: Emily

last: Troemel

department: Cell and Developmental Biology

institute: University of California, San Diego

description: Animals were synchronized by hypochlorite treatment and first larval stage (L1) arrest. Arrested L1s were placed onto NGM plates seeded with OP50 and grown at the permissive temperature (15°C) until L4, shifted to the restrictive temperature (25°C) for 6 hours and then harvested. daf-2;daf-16 animals are slightly younger than daf-2 animals in RepA.

hyb_protocol: Affymetrix

scan_protocol: Affymetrix

data_processing: Resolver Gene Expression Data Analysis System, version 5.1 (Rosetta Inpharmatics)

organism: Caenorhabditis elegans

label_protocol: Affymetrix

extract_protocol: TRI Reagent (Molecular Research Center)

tag: Strain

tag: Age

value: late L4/young adult

value: whole animal


title: daf2pmk1_RepB

description: Animals were synchronized by hypochlorite treatment and first larval stage (L1) arrest. Arrested L1s were placed onto NGM plates seeded with OP50 and grown at the permissive temperature (15°C) until L4, shifted to the restrictive temperature (25°C) for 6 hours and then harvested.


title: CHC357N-U133A


title: daf2pmk1_RepC

growth_protocol: Animals were synchronized by hypochlorite treatment and first larval stage (L1) arrest. Arrested L1s were placed onto NGM plates seeded with OP50 and grown at the permissive temperature (15°C) until L4, shifted to the restrictive temperature (25°C) for 6 hours and then harvested.


title: Young trifoliate leaf, biological replicate 2

status: Public on Apr 28 2007

city: Los Angeles

first: Bob

last: Goldberg

address: 610 Charles E Young Drive East

department: Molecular, Cell and Developmental Biology

institute: University of California, Los Angeles

description: frozen leaves were ground in liquid nitrogen using a mortar and pestle.

hyb_protocol: Following fragmentation, ten micrograms of cRNA were hybridized for 16 hr at 45¡C with the GeneChip Soybean Genome Array. GeneChips were washed and stained using the EukGE-WS2V4_450 protocol in the Affymetrix Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G System.

data_processing: The data were analyzed with the Affymetrix GeneChip Operating System 1.3 (GCOS 1.3) using default analysis settings and global scaling as a normalization method. The trimmed mean target intensity of each array was set to 500.

organism: Glycine max

source_name: Soybean leaf

label_protocol: Two nanograms of total RNA was amplified using the RiboAmp OA Amplification Kit (Molecular Devices, Sunnyvale, CA).  Biotinylated cRNAs were synthesized with the ENZO BioArray High Yield RNA Transcript Labeling Kit using all of the amplified RNA from the RiboAmp OA Kit.

growth_protocol: Soybean cv. Asgrow A3237 was grown in the UCLA Plant Growth Center using a 16:8 light-dark cycle at 22¡C.

extract_protocol: Total RNA was extracted using Plant RNA Isolation Reagent (Invitrogen, Carlsbad, CA).

treatment_protocol: Trifolate leaves (without pedicels) with lengths between 1.5 and 2.5 cm were collected, freezed in liquid nitrogen, and ground into powder with liquid nitrogen using a mortar and pestle.

tag: Soybean cv. Asgrow A3237


title: daf2daf16_RepB

description: Animals were synchronized by hypochlorite treatment and first larval stage (L1) arrest. Arrested L1s were placed onto NGM plates seeded with OP50 and grown at the permissive temperature (15°C) until L4, shifted to the restrictive temperature (25°C) for 6 hours and then harvested. daf-2;daf-16 animals are very slightly younger than daf-2 animals in RepB.


title: Young trifoliate leaf, biological replicate 1


title: 288_CA_1

extract_protocol: Total RNA of each one of the three reference components was isolated by Trizol, and treated with DNAse-I. Thereafter, mRNA of each component was linearly amplified for one round (MessageAmp aRNA kit, #1750, Ambion), sybthesizing antisense cRNAs.


title: daf2_RepC


title: daf2pmk1_RepA


title: daf2daf16_RepC

description: Animals were synchronized by hypochlorite treatment and first larval stage (L1) arrest. Arrested L1s were placed onto NGM plates seeded with OP50 and grown at the permissive temperature (15°C) until L4, shifted to the restrictive temperature (25°C) for 6 hours and then harvested. daf-2;daf-16 animals are slightly older than daf-2 animals in RepC.


title: daf2_RepB


title: daf2_RepA


title: Celegans_PA14_8hours_RepB

description: fer-15;fem-1 animals were synchronized by hypochlorite treatment and first larval stage (L1) arrest. Arrested L1s were placed onto NGM plates seeded with OP50 and grown at the restrictive temperature (25°C) in order to obtain sterile adults. After ~52 hours of growth at 25°C (young adulthood), animals were transferred to SK plates seeded either with OP50, wild-type PA14 or gacA mutant PA14. Animals were harvested at 4 hours and 8 hours after transfer.

treatment_protocol: Animals were transferred to P. aeruginosa PA14 for 8 hours prior to harvest.

value: young adult


title: Celegans_PA14_4hours_RepC

growth_protocol: fer-15;fem-1 animals were synchronized by hypochlorite treatment and first larval stage (L1) arrest. Arrested L1s were placed onto NGM plates seeded with OP50 and grown at the restrictive temperature (25°C) in order to obtain sterile adults. After ~52 hours of growth at 25°C (young adulthood), animals were transferred to SK plates seeded either with OP50, wild-type PA14 or gacA mutant PA14. Animals were harvested at 4 hours and 8 hours after transfer.

treatment_protocol: Animals were transferred to P. aeruginosa PA14 for 4 hours prior to harvest.


title: Celegans_PA14_8hours_RepC


title: Celegans_PA14_4hours_RepB


title: Celegans_gacA_8hours_RepB

treatment_protocol: Animals were transferred to P. aeruginosa PA14 mutant strain gacA for 8 hours prior to harvest.


title: Celegans_gacA_4hours_RepC

treatment_protocol: Animals were transferred to P. aeruginosa PA14 mutant strain gacA for 4 hours prior to harvest.


title: Celegans_gacA_8hours_RepA


title: Celegans_PA14_8hours_RepA


title: Celegans_PA14_4hours_RepA


title: Celegans_OP50_4hours_RepB

description: fer-15;fem-1 animals were synchronized by hypochlorite treatment and first larval stage (L1) arrest.  Arrested L1s were placed onto NGM plates seeded with OP50 and grown at the restrictive temperature (25°C) in order to obtain sterile adults.  After ~52 hours of growth at 25°C (young adulthood), animals were transferred to SK plates seeded either with OP50, wild-type PA14 or gacA mutant PA14.  Animals were harvested at 4 hours and 8 hours after transfer.

treatment_protocol: Animals were transferred to E. coli strain OP50 for 4 hours prior to harvest.


title: Celegans_gacA_8hours_RepC


title: Celegans_OP50_8hours_RepB

treatment_protocol: Animals were transferred to E. coli strain OP50 for 8 hours prior to harvest.


title: Celegans_OP50_8hours_RepA


title: 46855

status: Public on Feb 20 2006

city: Beltsville

first: Robert

middle: W

last: Li

state: MD

address: Barc East

department: Animal & Natural Resources Institute

institute: USDA

description: Single channel hybridization of biotin-labeled samples. The signal is detected using Cy3-streptavidin staining.

data_processing: quantile normalization (Bolstad et al., 2003).

organism: Rattus norvegicus

source_name: brain, cerebral cortex

tag: Each sequence is represented by 11 distinct 60mer oligos. The value is mean normalized intensity from all 11 oligos


title: Celegans_gacA_4hours_RepB


title: 55968

organism: Neospora caninum

source_name: a temperature-sensitive strain, ts-8, of Neospora caninum


title: Celegans_gacA_4hours_RepA


title: 46544

source_name: a temperature-sensitive strain of Neospora caninum, ts-8


title: Celegans_OP50_4hours_RepC

growth_protocol: fer-15;fem-1 animals were synchronized by hypochlorite treatment and first larval stage (L1) arrest.  Arrested L1s were placed onto NGM plates seeded with OP50 and grown at the restrictive temperature (25°C) in order to obtain sterile adults.  After ~52 hours of growth at 25°C (young adulthood), animals were transferred to SK plates seeded either with OP50, wild-type PA14 or gacA mutant PA14.  Animals were harvested at 4 hours and 8 hours after transfer.


title: 46867


title: 49589

source_name: wild type strain of Neospora caninum, NC-1


title: 47094


title: 64851


title: 289_LU_1

source_name: common reference


title: Celegans_OP50_8hours_RepC


title: 64349


title: PGA-Band_Sham_48h_15-m5

status: Public on Jul 10 2002

city: Cambridge

first: Martina

last: Schinke

state: MA

address: 7 Divinity Ave

department: Bauer Center for Genomic Research

institute: Harvard University

description:
Pressure-overload induced hypertrophy model.
Mouse was sacrificed 48 hours after sham operation.
Keywords = cardiac hypertrophy

source_name: Left heart ventricle


title: Celegans_OP50_4hours_RepA


title: 13_W.E.M.5, Experimental replicate W

source_name: Soybean cultivar OX20-8 - Mock Inoculated (Inoculated with water + agar): 120 hours post inoculation

tag: cultivar - OX20-8

tag: Tissue/Cell Type

tag: total Soybean RNA Plus 16 % P.sojae RNA added


title: Patient 437 Core Set (cancer panel)

status: Public on Jun 10 2008

city: Rochester

middle: B.

last: Jenkins

state: MN

address: 200 First Street SW

department: Laboratory Medicine and Pathology

institute: Mayo Clinic

hyb_protocol: hybridization was performed in accordance with the manufacturer's instructions

scan_protocol: Arrays were scanned using Illumina's BeadArray Reader

data_processing: Illumina's Bead Studio Software using no normalization was used to process the image files. The raw data was then normalized using cyclic loess (fastlo). The normalized value for the probes was averaged to determine the expression level for the gene. The DASL method labels each sample with a mixture of CY3 and Cy5. Only a single sample is applied to each array.  The scanner measures fluorescence of both Cy3 and Cy5.  This data is contained in the red and grn files for each sample. The bead studio software averages the red and green signals to provide a single value for the expression of a particular probe.

label: cy3, cy5

source_name: primary prostate cancer

label_protocol: cy3, cy5 labeling was performed in accordance with the manufacturer's instructions

extract_protocol: Four freshly cut 10 micron sections of FFPE tissue were deparaffinized and the Gleason dominant cancer focus was macrodissected. RNA was extracted using the High Pure RNA Paraffin Kit from Roche (Indianapolis, IN).

tag: Case-Control Group

value: PSA

tag: Training or Validation

value: trn

tag: Age (yrs) at RRP

tag: PSA (ng/ml) at RRP

tag: Revised Gleason Score

tag: Stage

value: TxN+

tag: Ploidy

value: tetraploid

tag: First Rising PSA (ng/ml)

tag: Time (yrs) from RRP to first rising PSA

tag: Second Rising PSA (ng/ml)

tag: Time (yrs) from RRP to second rising PSA

tag: PSA Slope (ng/ml/yr)

tag: Time (yrs) from PSA recurrence to systemic disease

tag: Years of Followup after RRP

tag: patient's status

value: Alive

tag: Prostate cancer specific death

value: No


title: Patient 275 Core Set (custom panel)

value: NED

value: diploid


title: Patient 237 Core Set (custom panel)

value: val