title: opsin KO-LL30-1

status: Public on Mar 09 2009

city: Hanover

first: Chen-Hui

last: Chen

state: NH

address: 702 Remsen

department: Genetics

institute: Dartmouth College

description: reference RNA design

hyb_protocol: Before prehybridization, microarray slides were pre-treated with 600 mJ UV and Pronto Background Reduction kit (Corning) according to the manufacturer’s protocol. Slides were prehybridized at 42C for 45 min with prehybridization buffer (5X SSC, 0.1% SDS and 1% BSA) and then spun dry for one min at 1000 RPM. The dried cDNA sample was rehydrated in 31.5 l hybridization buffer (40% formamide, 5X SSC, 0.1 SDS) for several minutes before adding 3.5 l 10X blocker (1µg/µl ssDNA and 2µg/µl tRNA in water, Sigma). The mixture was heated at 95 C for 3 min, cooled down on ice for 30 seconds and then quickly spun before pipetting onto the slide under a Lifter slip (VWR). The slide was placed in the oven (Boekel InSlide Out Model 241000 hybridization oven) for hybridization overnight at 42C. To maintain humidity in the hybridization cassette, 2 ml of hybridization buffer containing 40% formamide were pipetted onto 4 Whatman 42.5 mm filter paper circles within the cassette. To wash the slides, 2X SSC/ 0.1% SDS was used for the first incubation at 42C for 5 min and then followed by 0.1X SSC/ 0.1% SDS at RT for 10 min. The final wash was performed twice using 0.1X SSC only at RT for 5 min. Slides were spun dry before scanning.

scan_protocol: The slides were scanned with a GMS 418 microarray scanner (Affymetrix). After griding with ScanAlyze 2.51, the raw data were uploaded and normalized [Intensity based (global)] with GeneTraffic software (Stratagene).

data_processing: Spots of insufficient quality were flagged by visual inspection and signal to background noise ratio. Due to the fact that some light inducible genes have almost no transcription in the dark, only spots with a fluorescent intensity more than 1 fold in sample channel and more than 2 fold in reference channel over local/average background were selected for further analysis. Genes missing more than 25% of their measurements from 90 microarrays were all excluded, which results in 5588 valid spots, providing approximately 53.8% coverage for total 10372 spots on the array. In order to pick up genes with low expression, genes with at least 2 arrays with absolute values of log2 ratio greater than 0.5 were all included. On the other hand, only genes have more than 4 fold changes (maximum - minimum) across 90 microarrays were included for final clustering analysis. Totally, 2864 out of 10372 spots passed these selection criteria. Unsupervised hierarchical clustering was done using Cluster 3.0 with method of average linkage after mean centering Pearson correlation (uncentered). The graphical representations of clustering results were generated using TreeView 1.0.13.

organism: Neurospora crassa

source_name: nop knockout, LP, 30 min.

label_protocol: 5 μg anchored oligo(dT) 20mer (2.5µg/µl, Invitrogen) was mixed with the RNA sample in a total of 18.4 μl. After incubation at 70 ˚C for 10 min, the mixture was cooled on ice for another 10 min. Reverse transcription was performed using 400 U Superscript II Reverse Transcriptase (200 U/μl, Invitrogen) with amino-allyl-dUPT/dNTP (aadUPT: 15 mM, dTTP: 10 mM, dATP: 25 mM, dCTP: 25 mM, dGTP: 25 mM) at 42 ˚C for 2 hours in a 30 µl reaction volume. The RNA was then hydrolyzed by adding 10 l 1 M NaOH and 10 l 0.5 M EDTA. After incubation at 65 ˚C for 15 min, 25 l 1 M Tris pH 7.4 buffer was added to neutralize the reaction. The cDNA was then cleaned up with YM 30 column (Millipore). The eluted cDNA was dried in speed vacuum without heat for about 1-1.5 hour and then resuspend in 5 l H2O at RT for 15 min. 3 µl 25 mg/ml sodium bicarbonate buffer (Sigma) and 2 µl Alexa dye (Invitrogen) was added. The coupling reaction was incubated for 1 hour in the dark at RT and then inactivated by adding 4.5 µl 4 M hydroxylamine. After15 min in the dark at RT, the labeled cDNA was cleaned up using CyScribe GFX Purification Kit (Amersham) following the manufacturer’s protocol. cDNA was dried in speed vacuum without heat for another 1-1.5 hour. Alexa Fluor 555 was consistently used to label cDNA synthesized from reference RNA, which is a mixture containing equal amounts of RNA samples harvested from different circadian time points and light treatment durations. The same batch of pooled RNA was used as a reference for each array experiment. Alexa Fluor 647 was used exclusively to label cDNA representing sample RNA.

growth_protocol: Frozen conidia were inoculated onto a regular minimal slant one week in advance to generate fresh conidia. On day 0, conidia were suspended in sterile water for quantification. To form a mycelial layer, 1 x 107 of conidia or similar amount of mass (for knockout strain of NCU02713.2) were inoculated into a 10 cm Petri dish with 20 ml Bird medium containing 2% glucose. After 24 hours of incubation in darkness at 25˚C, a mycelial plug was cut with a size No.4 cork borer (8 mm diameter) and transferred into a 125 ml flask with 50 ml Bird medium containing 2% glucose. All procedures were performed under low red light environment to avoid any possible light-stimulating effects.

extract_protocol: Total RNA was extracted using TRIZOL reagent (Invitrogen) with a homogenizer (Tissuelyzer, Qiagen) containing stainless beads for pulverizing. Total RNA was then subjected to an additional purification step using RNeasy spin columns (Qiagen). After quantification by spectrophototmeter, 20 μg RNA from each time point was used for cDNA synthesis.

treatment_protocol: After another 24 hours of culture with constant shaking (125 rpm) in darkness at 25 ˚C, the flasks were moved to a shaker in LL at 25 ˚C with a continuous white light stimulus, covering wide-range of spectrum from 400 nm to 700 nm (cool white fluorescent light bulb, General Electric F20T12-CW, 40-50 μmole photons/m2/s) and then harvested after 5 (LL5), 10 (LL10), 15 (LL15), 30 (LL30), 45 (LL45), 60 (LL60), 90 (LL90), 120 (LL120), 240 (LL240) minutes of white light treatment. Using vacuum filtration, dried mycelia tissues were immediately frozen in liquid nitrogen and stored at -80˚C until RNA extraction. Control DD samples were harvested under low red light condition.

tag: mycelia from liquid culture

source_name: pooled RNA from 74A


title: cry KO-LL30-1

type: RNA

country: USA

description: reference RNA design

label: Alexa Fluor 647

molecule: total RNA

source_name: cry knockout, LP, 30 min.

label: Alexa Fluor 555


title: opsin KO-LL120-1

description: reference RNA design

source_name: nop knockout, LP, 120 min.


title: opsin KO-LL15-1

description: reference RNA design

source_name: nop knockout, LP, 15 min.


title: opsin KO-LL60-1

description: reference RNA design

source_name: nop knockout, LP, 60 min.


title: GM07048

status: Public on Mar 23 2009

city: Chicago

first: Eileen

last: Dolan

state: IL

address: 5841 S. Maryland

department: Medicine

institute: University of Chicago

description: Homo sapiens lymphoblastoid cell lines (30 CEPH trios and 30 Yoruban  trios) were purchased from Coriell Institute for Medical Reseach (Camden, NJ).

hyb_protocol: Approximately 5.5ug of labeled DNA target was hybridized to the Affymetrix GeneChip Human Exon 1.0 ST Array at 45° C for 16 hours per manufacturer's recommendation (see http://www.affymetrix.com/products/arrays/exon_application.affx for additional information).  Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450.

scan_protocol: All samples were scanned on a GCS3000 Scanner (Affymetrix, Inc.).

data_processing: Expression arrays were analyzed using the Affymetrix PowerTools v1.8.6 (http://www.affymetrix.com/support/developer/powertools/index.affx). The start and end coordinates of all probes represented on the exon array were queried and determined against the human genome (hg18). The coordinates for all SNPs were then queried in the dbSNP database (release 129) (http://www.ncbi.nlm.nih.gov/projects/SNP) and used to identify probes harboring SNPs.  Of the ~1.4 million probesets on the exon array, ~288,400 contained at least one probe with a SNP. The probeset signal intensity files were filtered by removing those ~288,400 probes from the probesets harboring these known SNPs. Probe intensities were then background corrected and quantile normalized over all 176 samples. The data were then log2 transformed with a median polish. Gene-level expression of 17,879 transcript clusters was summarized using the RMA (robust multi-array average) method with signals generated on a core set (i.e. with RefSeq-supported annotation) of exons (~288,400 probesets). A transcript cluster or probeset was defined to be reliably expressed in LCLs if the log2 transformed expression signal was greater than 5 in at least 80% of the 176 samples. 10,796 of the 17,879 core transcript clusters met these criteria(15). To avoid annotation ambiguity, the final analysis dataset is comprised of 7,860 expressed transcript clusters (corresponding to 115,544 probesets, a minimum of 3 probesets for each transcript cluster) with unique gene annotations (based on NCBI Human Genome Build 34) as retrieved at the Affymetrix NetAffx Ananlysis Center (http://www.affymetrix.com/analysis/index.affx).

label: biotin

organism: Homo sapiens

source_name: lymphoblastoid cell line

label_protocol: Ribosomal RNA was depleted from 1ug of total RNA using the RiboMinus Human/Mouse Transcriptome Isolation kit (Invitrogen Corp., Carlsbad, CA).  cDNA was generated using the GeneChip WT cDNA Synthesis and Amplification Kit (Affymetrix, Inc., Santa Clara, CA) per manufacturer's instructions.  cDNA was fragmented and end labeled using the GeneChip WT Terminal Labeling Kit (Affymetrix, Inc.).

growth_protocol: Lymphoblastoid cell lines were centrifuged at 400 × g to remove media and 5 mL fresh lymphoblastoid cell media (LCL media) containing RPMI 1640 (Mediatech)/1% l-glutamine (Mediatech) plus 20% FBS (HyClone Laboratories) for the initial passage and then passaged every 48 h with LCL medium and 15% FBS. Cell suspensions were transferred to 25 cm2 flasks and incubated at 37° C in a 90% humidified 5% CO2 atmosphere. Cell lines were maintained at a concentration of 3.5-4.0×105 cells/mL and harvested following the 4th passage, only if viability ≥ 85%.

extract_protocol: Lymphoblastoid cell line suspensions were spun at 400 × g for 5 min to remove media. Cell pellets were washed twice with ice-cold PBS and stored at -80° C. Total RNA was extracted using RNeasy Plus Kits according to the manufacturers protocol.

treatment_protocol: Each sample was collected while in exponential growth, all remained untreated.  Baseline expression data.

tag: Coriell cell line repository identifier

tag: CEPH sample

tag: Gender

value: Male

tag: Associated family

tag: Family relationship

value: father


title: GM18856

tag: Yoruban sample


title: vvd KO-LL120-1

description: reference RNA design

source_name: vvd knockout, LP, 120 min.


title: vvd KO-LL30-1

description: reference RNA design

source_name: vvd knockout, LP, 30 min.


title: opsin KO-DD-1

description: reference RNA design

source_name: nop knockout, DD, 0.


title: GM18854

value: child


title: GM18852

value: Female

value: mother


title: GM18521


title: vvd KO-LL15-1

description: reference RNA design

source_name: vvd knockout, LP, 15 min.


title: cry KO-LL15-1

description: reference RNA design

source_name: cry knockout, LP, 15 min.


title: vvd KO-DD-1

description: reference RNA design

source_name: vvd knockout, DD, 0.


title: GM06985

value: maternal grandmother


title: GM18853


title: vvd KO-LL60-1

description: reference RNA design

source_name: vvd knockout, LP, 60 min.


title: cry KO-LL60-1

description: reference RNA design

source_name: cry knockout, LP, 60 min.


title: cry KO-LL120-1

description: reference RNA design

source_name: cry knockout, LP, 120 min.


title: cry KO-DD-1

description: reference RNA design

source_name: cry knockout, DD, 0.


title: T5 KO-LL15-1

description: reference RNA design

source_name: NCU03643.2 knockout, LP, 15 min.


title: T5 KO-LL120-1

description: reference RNA design

source_name: NCU03643.2 knockout, LP, 120 min.

source_name: pooled RNA from 74A


title: T5 KO-LL30-1

description: reference RNA design

source_name: NCU03643.2 knockout, LP, 30 min.


title: T5 KO-LL60-1

description: reference RNA design

source_name: NCU03643.2 knockout, LP, 60 min.


title: T5 KO-DD-1

description: reference RNA design

source_name: NCU03643.2 knockout, DD, 0.


title: T3 KO-LL60-1

description: reference RNA design

source_name: sah-1 knockout, LP, 60 min.


title: T2 KO-LL15-1

description: reference RNA design

source_name: NCU02713.2 knockout, LP, 15 min.


title: T3 KO-DD-1

description: reference RNA design

source_name: sah-1 knockout, DD, 0.


title: T4 KO-LL30-1

description: reference RNA design

source_name: vad-3 knockout, LP, 30 min.


title: T3 KO-LL15-1

description: reference RNA design

source_name: sah-1 knockout, LP, 15 min.


title: T4 KO-LL60-1

description: reference RNA design

source_name: vad-3 knockout, LP, 60 min.


title: T2 KO-LL30-1

description: reference RNA design

source_name: NCU02713.2 knockout, LP, 30 min.


title: T4 KO-LL120-1

description: reference RNA design

source_name: vad-3 knockout, LP, 120 min.


title: T3 KO-LL30-1

description: reference RNA design

source_name: sah-1 knockout, LP, 30 min.


title: T3 KO-LL120-1

description: reference RNA design

source_name: sah-1 knockout, LP, 120 min.


title: T2 KO-LL120-1

description: reference RNA design

source_name: NCU02713.2 knockout, LP, 120 min.


title: T1 KO-LL30-1

description: reference RNA design

source_name: sub-1 knockout, LP, 30 min.


title: T4 KO-DD-1

description: reference RNA design

source_name: vad-3 knockout, DD, 0.


title: T1 KO-LL120-1

description: reference RNA design

source_name: sub-1 knockout, LP, 120 min.

source_name: pooled RNA from 74A


title: T1 KO-LL15-1

description: reference RNA design

source_name: sub-1 knockout, LP, 15 min.


title: T2 KO-LL60-1

description: reference RNA design

source_name: NCU02713.2 knockout, LP, 60 min.


title: T4 KO-LL15-1

description: reference RNA design

source_name: vad-3 knockout, LP, 15 min.


title: T1 KO-LL60-1

description: reference RNA design

source_name: sub-1 knockout, LP, 60 min.


title: T1 KO-DD-1

description: reference RNA design

source_name: sub-1 knockout, DD, 0.


title: 74A-LL30-2

description: reference RNA design

source_name: 74A(wild-type), LP, 30 min.


title: 74A-LL60-2

description: reference RNA design

source_name: 74A(wild-type), LP, 60 min.


title: GM19207

source_name: lymphoblastoid cell line


title: 74A-LL120-2

description: reference RNA design

source_name: 74A(wild-type), LP, 120 min.


title: T2 KO-DD-1

description: reference RNA design

source_name: NCU02713.2 knockout, DD, 0.


title: wc-1, wc-2 KO-LL90-1

description: reference RNA design

source_name: wc-1, wc-2 knockout, LP, 90 min.


title: 74A-DD60-1

description: reference RNA design

source_name: 74A(wild-type), DD, 60 min.


title: wc-1, wc-2 KO-LL120-1

description: reference RNA design

source_name: wc-1, wc-2 knockout, LP, 120 min.


title: 74A-DD120-1

description: reference RNA design

source_name: 74A(wild-type), DD, 120 min.


title: 74A-DD5-1

description: reference RNA design

source_name: 74A(wild-type), DD, 5 min.


title: 74A-DD90-1

description: reference RNA design

source_name: 74A(wild-type), DD, 90 min.


title: 74A-DD240-1

description: reference RNA design

source_name: 74A(wild-type), DD, 240 min.

source_name: pooled RNA from 74A


title: wc-1, wc-2 KO-LL240-1

description: reference RNA design

source_name: wc-1, wc-2 knockout, LP, 240 min.


title: wc-1, wc-2 KO-LL15-1

description: reference RNA design

source_name: wc-1, wc-2 knockout, LP, 15 min.


title: 74A-DD-2

description: reference RNA design

source_name: 74A(wild-type), DD, 0.


title: wc-1, wc-2 KO-LL5-1

description: reference RNA design

source_name: wc-1, wc-2 knockout, LP, 5 min.


title: 74A-DD15-1

description: reference RNA design

source_name: 74A(wild-type), DD, 15 min.


title: wc-1, wc-2 KO-LL30-1

description: reference RNA design

source_name: wc-1, wc-2 knockout, LP, 30 min.


title: wc-1, wc-2 KO-DD-1

description: reference RNA design

source_name: wc-1, wc-2 knockout, DD, 0.


title: wc-1, wc-2 KO-LL60-1

description: reference RNA design

source_name: wc-1, wc-2 knockout, LP, 60 min.


title: wc-1, wc-2 KO-LL45-1

description: reference RNA design

source_name: wc-1, wc-2 kncokout, LP, 45 min.


title: 74A-LL15-2

description: reference RNA design

source_name: 74A(wild-type), LP, 15 min.


title: wc-2 KO-LL240-1

description: reference RNA design

source_name: wc-2 knockout, LP, 240 min.


title: 74A-DD30-1

description: reference RNA design

source_name: 74A(wild-type), DD, 30 min.


title: wc-1, wc-2 KO-LL10-1

description: reference RNA design

source_name: wc-1, wc-2 knockout, LP, 10 min.


title: wc-2 KO-LL90-1

description: reference RNA design

source_name: wc-2 knockout, LP, 90 min.


title: 74A-DD45-1

description: reference RNA design

source_name: 74A(wild-type), DD, 45 min.


title: 74A-DD-3

description: reference RNA design


title: wc-2 KO-LL120-1

description: reference RNA design

source_name: wc-2 knockout, LP, 120 min.


title: wc-2 KO-LL60-1

description: reference RNA design

source_name: wc-2 knockout, LP, 60 min.

source_name: pooled RNA from 74A


title: wc-2 KO-LL5-1

description: reference RNA design

source_name: wc-2 knockout, LP, 5 min.


title: wc-2 KO-LL10-1

description: reference RNA design

source_name: wc-2 knockout, LP, 10 min.


title: 74A-DD10-1

description: reference RNA design

source_name: 74A(wild-type), DD, 10 min.


title: wc-2 KO-LL45-1

description: reference RNA design

source_name: wc-2 kncokout, LP, 45 min.


title: wc-2 KO-LL15-1

description: reference RNA design

source_name: wc-2 knockout, LP, 15 min.


title: wc-2 KO-DD-1

description: reference RNA design

source_name: wc-2 knockout, DD, 0.


title: wc-1 KO-LL240-1

description: reference RNA design

source_name: wc-1 knockout, LP, 240 min.


title: wc-2 KO-LL30-1

description: reference RNA design

source_name: wc-2 knockout, LP, 30 min.


title: wc-1 KO-LL90-1

description: reference RNA design

source_name: wc-1 knockout, LP, 90 min.


title: frq7-LL45-1

description: reference RNA design


title: wc-1 KO-DD-1

description: reference RNA design

source_name: wc-1 knockout, DD, 0.


title: frq7-LL60-1

description: reference RNA design


title: frq7-LL90-1

description: reference RNA design


title: frq7-LL120-1

description: reference RNA design


title: wc-1 KO-LL120-1

description: reference RNA design

source_name: wc-1 knockout, LP, 120 min.


title: frq7-LL30-1

description: reference RNA design


title: wc-1 KO-LL10-1

description: reference RNA design

source_name: wc-1 knockout, LP, 10 min.


title: wc-1 KO-LL5-1

description: reference RNA design

source_name: wc-1 knockout, LP, 5 min.


title: frq7-LL240-1

description: reference RNA design


title: wc-1 KO-LL15-1

description: reference RNA design

source_name: wc-1 knockout, LP, 15 min.


title: wc-1 KO-LL30-1

description: reference RNA design

source_name: wc-1 knockout, LP, 30 min.


title: wc-1 KO-LL45-1

description: reference RNA design

source_name: wc-1 kncokout, LP, 45 min.


title: frq7-LL15-1

description: reference RNA design

source_name: pooled RNA from 74A


title: phy-1, phy-2  KO-LL120-1

description: reference RNA design

source_name: phy-1, phy-2 knockout, LP, 120 min.


title: frq7-LL5-1

description: reference RNA design


title: wc-1 KO-LL60-1

description: reference RNA design

source_name: wc-1 knockout, LP, 60 min.


title: frq7-LL10-1

description: reference RNA design


title: frq7-DD-1

description: reference RNA design


title: phy-1, phy-2  KO-LL240-1

description: reference RNA design

source_name: phy-1, phy-2 knockout, LP, 240 min.


title: phy-1, phy-2  KO-LL60-1

description: reference RNA design

source_name: phy-1, phy-2 knockout, LP, 60 min.


title: phy-1, phy-2  KO-LL30-1

description: reference RNA design

source_name: phy-1, phy-2 knockout, LP, 30 min.


title: phy-1, phy-2  KO-LL5-1

description: reference RNA design

source_name: phy-1, phy-2 knockout, LP, 5 min.


title: phy-1, phy-2  KO-LL90-1

description: reference RNA design

source_name: phy-1, phy-2 knockout, LP, 90 min.


title: phy-2 KO-LL90-1

description: reference RNA design

source_name: phy-2 knockout, LP, 90 min.


title: phy-1, phy-2  KO-LL15-1

description: reference RNA design

source_name: phy-1, phy-2 knockout, LP, 15 min.


title: phy-2 KO-LL120-1

description: reference RNA design

source_name: phy-2 knockout, LP, 120 min.


title: phy-1, phy-2  KO-LL45-1

description: reference RNA design

source_name: phy-1, phy-2 kncokout, LP, 45 min.


title: phy-2 KO-LL240-1

description: reference RNA design

source_name: phy-2 knockout, LP, 240 min.


title: phy-1, phy-2  KO-LL10-1

description: reference RNA design

source_name: phy-1, phy-2 knockout, LP, 10 min.


title: phy-2 KO-DD-1

description: reference RNA design

source_name: phy-2 knockout, DD, 0.


title: phy-2 KO-LL30-1

description: reference RNA design

source_name: phy-2 knockout, LP, 30 min.


title: phy-2 KO-LL5-1

description: reference RNA design

source_name: phy-2 knockout, LP, 5 min.


title: phy-1 KO-LL120-1

description: reference RNA design

source_name: phy-1 knockout, LP, 120 min.

source_name: pooled RNA from 74A


title: phy-1 KO-LL10-1

description: reference RNA design

source_name: phy-1 knockout, LP, 10 min.


title: phy-1, phy-2  KO-DD-1

description: reference RNA design

source_name: phy-1, phy-2 knockout, DD, 0.


title: phy-1 KO-LL5-1

description: reference RNA design

source_name: phy-1 knockout, LP, 5 min.


title: phy-1 KO-LL15-1

description: reference RNA design

source_name: phy-1 knockout, LP, 15 min.


title: phy-1 KO-LL60-1

description: reference RNA design

source_name: phy-1 knockout, LP, 60 min.


title: phy-1 KO-LL90-1

description: reference RNA design

source_name: phy-1 knockout, LP, 90 min.


title: phy-2 KO-LL60-1

description: reference RNA design

source_name: phy-2 knockout, LP, 60 min.


title: phy-1 KO-LL240-1

description: reference RNA design

source_name: phy-1 knockout, LP, 240 min.


title: phy-2 KO-LL10-1

description: reference RNA design

source_name: phy-2 knockout, LP, 10 min.


title: phy-2 KO-LL15-1

description: reference RNA design

source_name: phy-2 knockout, LP, 15 min.


title: phy-1 KO-DD-1

description: reference RNA design

source_name: phy-1 knockout, DD, 0.


title: phy-1 KO-LL30-1

description: reference RNA design

source_name: phy-1 knockout, LP, 30 min.


title: 74A-LL120-1

description: reference RNA design

source_name: 74A(wild-type), LP, 120 min.


title: 74A-LL5-1

description: reference RNA design

source_name: 74A(wild-type), LP, 5 min.


title: 74A-LL10-1

description: reference RNA design

source_name: 74A(wild-type), LP, 10 min.


title: 74A-LL45-1

description: reference RNA design

source_name: 74A(wild-type), LP, 45 min.


title: 74A-LL30-1

description: reference RNA design

source_name: 74A(wild-type), LP, 30 min.

source_name: pooled RNA from 74A


title: 74A-LL240-1

description: reference RNA design

source_name: 74A(wild-type), LP, 240 min.


title: 74A-LL60-1

description: reference RNA design

source_name: 74A(wild-type), LP, 60 min.


title: 74A-LL15-1

description: reference RNA design

source_name: 74A(wild-type), LP, 15 min.


title: 74A-DD-1

description: reference RNA design

source_name: 74A(wild-type), DD, 0.


title: 74A-LL90-1

description: reference RNA design

source_name: 74A(wild-type), LP, 90 min.


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h1258)

status: Public on Mar 04 2009

city: Cleveland

first: Peter

middle: A

last: Wilkinson

state: Ohio

address: 2103 Cornell Road

department: Pathology / Systems Biology & Bioinformatics

institute: Case Western Reserve University

description: Peripheral blood mononuclear cells (PBMCs) from healthy adult individuals were isolated by Ficoll-HyPaque (Pharmacia) density gradient. We first enriched for CD4+ T cells using negative immunomagnetic beads selection (Automacs, Myltenii), cells were then labeled with anti-CD4-APCcy7, anti-CD45RA-ECD, anti-CD27-FITC and anti-CCR7-PEcy7 and sorted into Naïve cells described as CD4+, CD45RA+, CD27+ and CCR7+, Central Memory cells (TCM) described as CD4+, CD45RA-, CD27+ and CCR7+ cells and Effector Memory cells (TEM) described as CD4+, CD45RA-, CD27- and CCR7- cells. Sorting was performed using a BDAria (BD Pharmingen).  Purity of the TCM and TEM sub-populations was ranging from 96 to 99%. All procedures were done at 4C to avoid any changes in cell phenotype or gene expression.

hyb_protocol: similar to direct labelling protocol lists at UHN Microarray facility web site.

scan_protocol: Scanned at 16 bits using the ScanArray Express Scanner (Packard Bioscience) at 10micro-meter resolution at 635 nm (Cy5) and 532 nm (Cy3) using Genepix Pro 6.0 analysis software (Molecular Divices Corporation)

data_processing: Each quantified output file was run though the following pre-processing steps using R (URL: www.r-project.org)and the Limma package. For Minimum Intensity filtering, R and G values were treated with a surrogate replacement policy for estimating sub-threshold values. For normalization within Arrays, the raw merged R and G channels were lowess-normalized (grouped by print-tip) and transformed to log2 ratios (70).

source_name: Universal RNA

label_protocol: Universal RNA probes was prepared by direct labeling of universal aRNA with Cy-3 fluorescent dye

extract_protocol: Universal human reference RNA was purchased from Stratagene and was amplified using MessageAMP aRNA kits (Ambion) as per manufacturer's instructions prior to labeling

tag: Tissue:Universal RNA

source_name: PBMC from Successful Treated (ST) donor

label_protocol: Sample probes were prepared by direct labeling with Cy-5 fluorescent dye

extract_protocol: Total RNA was isolated from beads purified CD4+T cell populations using an RNA extraction kit (Quiagen) then amplified using the MessageAmp RNA kit (Ambion) as per the manufacturer's instructions.


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h1307)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h1309)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mEFMs0h1137)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mCEMs0h1070)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h1259)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h1149)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h1083)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mEFMs0h1132)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h1059)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h1082)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mCEMs0h1136)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h1058)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h1045)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mEFMs0h1071)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h1148)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h1046)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h0962)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h0963)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h1035)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h1034)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h0957)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h0956)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mCEMs0h1016)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mCEMs0h0970)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mEFMs0h0999)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mEFMs0h1017)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h0944)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h0947)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h0952)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mEFMs0h0953)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h0949)


title: PBMC from Successful Treated (ST) donor (pAVIRcCD4mCEMs0h0954)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mEFMs0h0923)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mEFMs0h0884)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mCEMs0h0883)


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mCEMs0h0922)


title: Ovary Babesia Infected (6 hours) B. microplus

status: Public on Mar 01 2009

type: SAGE

anchor: NlaIII

city: Kerrville

first: Leonel

middle: (Leo)

last: Saldivar

state: TX

address: 2700 Fredericksburg Rd.

department: Agriculture

institute: USDA

description:
LongSAGE dataset, source material from B. microplus ovary tissue.   Tissue sample aquired from USDA-ARS Pullman WA laboratory.  This library consists of the babesia infected ovary tissue, i.e. at time 6 hours.
Supplemental files: include the multiple fasta files containing the concatemers that contain the LongSAGE tags, i.e. raw data.

data_processing:
The submitted LongSAGE library was constructed using perl scripts written by Leonel Saldivar (leo.saldivar@ars.usda.gov or leo.sal@gmail.com).   It extracted 17bp tags and did NOT remove duplicate ditags.      Script description below:
Short summary:    Given a fasta file containing multiple sequences representing LongSAGE concatemers containing putative 17mer LongSAGE tags, the script will extract the tags and construct a library from them, i.e. tags followed
by their respective counts. e.g.
tag1	24
tag2	2
Technical details:
Since I am looking for 17mer tags, the ditags of 34nt length are found but
also the ditags of 33, and 32 nt are extracted.  SAGE2000 (invitrogen's)
software extracts 32-34 ditags if the 17mer tags are selected from the menu.
The reasoning I believe is that if it is 33nt long there is one base that
overlaps both 17mer tags, in the case of the 32nt ditags, two bases are
shared (overlap).    I will do the same.   One tag is removed from the 5'
side while the other is extracted from the 3' end and reverse complemented.

organism: Rhipicephalus microplus

source_name: B. microplus Adult ovary tissue

extract_protocol: total RNA extracted using ToTALLY RNA from Ambion.

treatment_protocol: These ticks are the treatment in a babesia infected experiement.   The accompanying library includes the longSAGE counts for the control.

tag: B. microplus adult ovary tissue that was infected with babesia (6 hours)


title: Ovary control(time zero) B. microplus

description:
LongSAGE dataset, source material from B. microplus ovary tissue.   Tissue sample aquired from USDA-ARS Pullman WA laboratory.  This library consists of the control ovary tissue, i.e. at time zero, no Babesia infection introduced.
Supplemental files: include the multiple fasta files containing the concatemers that contain the LongSAGE tags, i.e. raw data.

source_name: Adult Ovary [B. microplus]

extract_protocol: total RNA was extracted from Adult B. microplus ovary tissue using the ToTALLY RNA kit from Ambion.

treatment_protocol: These ticks are the control in a babesia infected experiement.   The accompanying library will include the longSAGE counts 6 hours after infection.

tag: Sample

value: Collected at USDA-ARS lab Pullman, WA on Feb 23-25, 2005.

tag: Extraction protocol

value: ToTALLY RNA kit by Ambion.

tag: Ticks collected and dissected at a later date.


title: PBMC from Long Term Non Progressors (LTNP) donor (pLTNPcCD4mEFMs0h0762)


title: Soy_shoot apical meristem2

status: Public on Feb 20 2009

city: Parkville

first: Chui

middle: E

last: WONG

state: Victoria

address: University of Melbourne

department: Land and Food

country: Australia

institute: University of Melbourne

description: Two independent biological replicates were used in the experiment

hyb_protocol: cDNA labelling and Affymetrix Soybean GeneChip hybridization was carried out by AGRF (Australian Genome Research Facility, Melbourne, Australia) using 3 µg of total RNA.

scan_protocol: Carried out by AGRF according to standard protocol at www.affymetrix.com

data_processing: Expression levels were estimated from Affymetrix hybridization intensity data using MicroArray Suite 5.0 (Affymetrix 2001). Raw numeric values representing the signal of each feature were imported into AffylmGUI (Affymetrix linear modeling Graphical User Interface; Wettenhall & Smyth, 2004) for linear modeling of the microarray data and for identifying differentially expressed genes in SAMs. The data was normalized using Robust Multiarray Averaging (RMA) method and a linear model was then used to average data between replicate arrays and to look for variability between them (Wettenhall & Smyth, 2004). The list of transcripts that were detected to be differentially expressed at adjusted p-value of <0.01 was then filtered using a relatively conservative selection method. We applied a cut-off of a 2-fold ratio of expression and filtered out those genes with expression levels that were called as “Marginal” or “Absence” in all six arrays according to the Affymetrix Statistical Algorithms. Hybridization quality was also tested by checking the expression level of the soybean housekeeping control genes that include 18S rRNA, Actin, GSTa, cytochrome P450, SBP, Ubiquitin, and GAPDH. The expression ratios (SAM/NM) of the control genes were consistently in the range of 0.83–1.29.

label: Biotin

organism: Glycine max

source_name: Microdissected shoot apical mersitem

extract_protocol: RNA extracted using Qiagen RNAEasy Mini Kit

tag: Cultivar Bragg. 10-day-old plants grown under the greenhouse condition in the University of Melbourne


title: Soy_non-shoot apical meristem2

source_name: non-apical- meristem (NM) tissue consisted of primary stem, primary roots and mature leaves

label_protocol: cDNA labelling and Affymetrix Soybean GeneChip hybridization was carried out by AGRF (Australian Genome Research Facility, Melbourne, Australia) using 3 µg of total RNA.


title: ChIP-on-chip for anti-acetylated H4 products of failed rat heart

status: Public on Feb 19 2009

type: genomic

city: Shimotsukeshi

first: Hiroyuki

last: Mano

state: Tochigi

address: 3311-1 Yakushiji

department: Division of Functional Genomics

country: Japan

institute: Jichi Medical University

description: Anti-acetylated H4 ChIP products of Dahl rat with a failed heart was hybridized to Rat Genome 230 2.0 arrays.

hyb_protocol: Hybridized for 16 hr at 48C, and processed with the protocols for Affymetrix Encode arrays.

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

data_processing: The mean signal intenstiy for all probe sets were set to be 500 units.

molecule: genomic DNA

organism: Rattus norvegicus

source_name: 19w Dahl rat

label_protocol: DNA was fragmented, amplified with T7 RNA polymerase by in vitro trasncription, and labeled by Affymetrix labeling kit.

extract_protocol: Genomic DNA was isolated by chromatin immunoprecipitation for acetylated H4.

tag: LV myocytes isolated from Dahl rat with a failed heart


title: ChIP-on-chip for anti-dimethylated K27 products of failed rat heart

description: Anti-dimethylated K27 ChIP products of Dahl rat with a failed heart was hybridized to Rat Genome 230 2.0 arrays.

extract_protocol: Genomic DNA was isolated by chromatin immunoprecipitation for dimethylated K27.


title: Soybean_nonapicalmeristem

scan_protocol: Using standard protocol as recommended by www.affymetrix.com and was done at AGRF (Australian Genome Research Facility, Melbourne, Australia)

source_name: non-apical- meristem (NM) tissue consisted of primary stem, primary roots and mature leaves from 10-day-old soybean plant

extract_protocol: Qiagen RNeasy Mini Kit was used for all RNA extraction

tag: Cultivar Bragg, grown for 10 days after sowing under the greenhouse condition in the University of Melbourne


title: ChIP-on-chip for anti-dimethylated K4 products of normal rat heart

description: Anti-dimethylated K4 ChIP products of Dahl rat with a normal heart was hybridized to Rat Genome 230 2.0 arrays.

extract_protocol: Genomic DNA was isolated by chromatin immunoprecipitation for dimethylated K4.

tag: LV myocytes isolated from Dahl rat with a normal heart


title: ChIP-on-chip for anti-dimethylated K27 products of normal rat heart

description: Anti-dimethylated K27 ChIP products of Dahl rat with a normal heart was hybridized to Rat Genome 230 2.0 arrays.


title: ChIP-on-chip for anti-acetylated H3 products of failed rat heart

description: Anti-acetylated H3 ChIP products of Dahl rat with a failed heart was hybridized to Rat Genome 230 2.0 arrays.

extract_protocol: Genomic DNA was isolated by chromatin immunoprecipitation for acetylated H3.


title: ChIP-on-chip for anti-dimethylated K4 products of failed rat heart

description: Anti-dimethylated K4 ChIP products of Dahl rat with a failed heart was hybridized to Rat Genome 230 2.0 arrays.


title: ChIP-on-chip for anti-dimethylated K9 products of failed rat heart

description: Anti-dimethylated K9 ChIP products of Dahl rat with a failed heart was hybridized to Rat Genome 230 2.0 arrays.

extract_protocol: Genomic DNA was isolated by chromatin immunoprecipitation for dimethylated K9.


title: ChIP-on-chip for anti-trimethylated K20 products of failed rat heart

description: Anti-trimethylated K20 ChIP products of Dahl rat with a failed heart was hybridized to Rat Genome 230 2.0 arrays.

extract_protocol: Genomic DNA was isolated by chromatin immunoprecipitation for trimethylated K20.


title: ChIP-on-chip for anti-trimethylated K9 products of normal rat heart

description: Anti-trimethylated K9 ChIP products of Dahl rat with a normal heart was hybridized to Rat Genome 230 2.0 arrays.

extract_protocol: Genomic DNA was isolated by chromatin immunoprecipitation for trimethylated K9.


title: ChIP-on-chip for anti-trimethylated K9 products of failed rat heart

description: Anti-trimethylated K9 ChIP products of Dahl rat with a failed heart was hybridized to Rat Genome 230 2.0 arrays.


title: Soy_shoot apical meristem

tag: Cultivar Bragg. 10-day-old plants grown under the greenhouse condition in the University of Melbourne.


title: ChIP-on-chip for anti-dimethylated K9 products of normal rat heart

description: Anti-dimethylated K9 ChIP products of Dahl rat with a normal heart was hybridized to Rat Genome 230 2.0 arrays.


title: ChIP-on-chip with anti-H3 acetylation

description: Anti-acetyalted H3 ChIP products of Dahl rat with a normal heart was hybridized to Rat Genome 230 2.0 arrays.


title: Pre-IP genomic DNA of normal rat heart

description: Pre-IP gemomic DNA of Dahl rat with a normal heart was hybridized to Rat Genome 230 2.0 arrays.

hyb_protocol: Hybridized for 16 hr at 48C on GeneChip Human Genome Array, and processed with the protocols of Affymetrix Encode arrays.

extract_protocol: Genomic DNA was isolated by a standard protocol


title: ChIP-on-chip for anti-trimethylated K4 products of failed rat heart

description: Anti-trimethylated K4 ChIP products of Dahl rat with a failed heart was hybridized to Rat Genome 230 2.0 arrays.

extract_protocol: Genomic DNA was isolated by chromatin immunoprecipitation for trimethylated K4.


title: ChIP-on-chip for anti-acetylated H4 products of normal rat heart

description: Anti-acetyalted H4 ChIP products of Dahl rat with a normal heart was hybridized to Rat Genome 230 2.0 arrays.

source_name: Pre-IP genomic DNA of normal rat heart


title: ChIP-on-chip for anti-trimethylated K20 products of normal rat heart

description: Anti-trimethylated K20 ChIP products of Dahl rat with a normal heart was hybridized to Rat Genome 230 2.0 arrays.


title: ChIP-on-chip for anti-trimethylated K4 products of normal rat heart

description: Anti-trimethylated K4 ChIP products of Dahl rat with a normal heart was hybridized to Rat Genome 230 2.0 arrays.


title: phy-2 KO-LL45-1

description: reference RNA design

source_name: phy-2 kncokout, LP, 45 min.

extract_protocol: Total RNA was extracted using TRIZOL reagent (Invitrogen) with a homogenizer (Tissuelyzer, Qiagen) containing stainless beads for pulverizing. Total RNA was then subjected to an additional purification step using RNeasy spin columns (Qiagen). After quantification by spectrophototmeter, 20 μg RNA from each time point was used for cDNA synthesis.

tag: mycelia from liquid culture


title: phy-1 KO-LL45-1

description: reference RNA design

source_name: phy-1 kncokout, LP, 45 min.


title: mRNA quantitation of failed heart

description: LV myocytes of CHF rat was hybridized.

hyb_protocol: 2 ug of cDNA were hybridized for 18 hr at 45C on GeneChip Human Genome Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.

data_processing: The mean value for the internal control probe sets (defined by Affymetrix) was set to be 500.

source_name: 19 weeks Dahl rat with a high-sodium diet

label_protocol: Biotinylated cDNA were prepared according to the Ovation Biotin RNA Amplification and Labeling System, NuGEN Technologies, Inc.

extract_protocol: Total RNA was extracted by RNeasy mini spin column (Qiagen, Inc).


title: mRNA quantitation of normal rat heart

description: LV myocytes of normal rat was hybridized.


title: Cortical vs Spinal Cord NS (15136)

status: Public on Feb 15 2009

city: Los Angeles

first: Terry

last: Kelly

state: CA

address: 700 Westwood Blvd

institute: UCLA

description: as per agilent protocol

hyb_protocol: as per agilent protocol

scan_protocol: as per agilent protocol

data_processing: feature extraction from agilent

organism: Mus musculus

source_name: Cortical NS

label_protocol: Agilent Low input amplification

extract_protocol: Trizol

tag: E14 secondary NS from cd1 mouse

source_name: Spinal Cord NS


title: CTB-1

status: Public on Jan 22 2009

city: Vienna

first: Martin

last: Bilban

country: Austria

institute: Medical University of Vienna

description: Affymetrix

hyb_protocol: Affymetrix

scan_protocol: Affymetrix

data_processing: Affymetrix MAS5.0

source_name: First trimester cytotrophoblast cells grown in culture for 24 hours

label_protocol: Affymetrix

extract_protocol: trizol and RNeasy Mini kit

tag: First trimester placental villous material was digested with a 0.125% (v/v) trypsin solution (GIBCO, Life Technologies, Paisley, UK). The released cells were loaded on top of a Percoll (Pharmacia, Uppsala, Sweden) gradient ranging from 70 to 10% (v/v). After centrifugation, the band containing the trophoblasts was collected and sequentially purified using immunomagnetic beads (Dynabeads M-450, Dynal) conjugated to CD45 RB leukocyte common antigen (Dako, CA) and to fibroblast specific antibody A 502 (Dianova, Hamburg).

tag: Trophoblasts were cultured for 24 h in DMEM (GIBCO)

tag: with 10% FCS, 100 mg/ml streptomycin and 100 IU/ml penicillin

tag: (GIBCO).


title: EVT-3

description: Affymetrix

source_name: First trimester placental organ explants grown on matrigel

tag: Dissected mesenchymal villous tissue of early pregnancy was arranged radially on 80 μl drops of growth-factor-reduced Matrigel™ (BD, Bedford, MA) in 24 well plates (SFM; DMEM:Ham's F12 (Gibco) 1:1, supplemented with 100 µg/ml streptomycin/penicillin (Gibco) and incubated for 4 hours at 37°C, 5% CO2 to allow attachment. Wells were then carefully flooded with 500 µl SFM, incubated at 37°C, 5% CO2 and subsequent growth characteristics were monitored by light microscopy. Total RNA or tissue sections were  prepared after 72 hours of incubation.


title: Cortical vs Spinal Cord NS (16178)

description: as per agilent protocol


title: Cortical vs Spinal Cord NS (16177)

description: as per agilent protocol


title: Cortical vs Spinal Cord NS

description: as per agilent protocol

tag: E14 Secondary NS from cd1 mouse


title: EVT-1

description: Affymetrix


title: EVT-2

description: Affymetrix


title: Pre-IP genomic DNA of failed rat heart

description: Pre-IP gemomic DNA of Dahl rat with a failed heart was hybridized to Rat Genome 230 2.0 arrays.

hyb_protocol: Hybridized for 16 hr at 48C, and processed with the protocols of Affymetrix Encode arrays.

source_name: 19 week old Dahl rat


title: CTB-2

description: Affymetrix

source_name: First trimester cytotrophoblast cells grown in culture for 24 hours.


title: Cortical vs Spinal Cord NS (14135)

description: as per agilent protocol


title: prostate_cancer_16

status: Public on Jan 21 2009

city: Pozuelo de Alarcon

first: Jose M.

last: Arencibia

state: Madrid

department: Biochemistry and Biotechnology

country: Spain

institute: Universidad Francisco de Vitoria

description: Human prostate cancer hybridized against normal adjacent prostate tissue from the same patient

hyb_protocol: Concentrated fluorescent cDNA probes (Cy3 and Cy5) were combined, ethanol precipitated and resuspended in 40 microlitres of SlideHyb Buffer #1 (Ambion). Ten micrograms of polyA (SIGMA) and 4 micrograms of yeast tRNA (GIBCO) were added as blocking agents and the samples were denatured at 95ºC for 2-3 minutes and applied to the prehybridized microarray slide. Prehybridization of microarray slides was performed in 4x SSC, 0.1% SDS and 0.002% BSA for 45 minutes at 65ºC. Hibridization was carried out at 42ºC for 15-20 hours. After hibridization, slides were washed 3 times in 2X SSC, 0.5 % SDS for 15 minutes each at 42ºC and then 3 times in 0.5X SSC, 0.5 % SDS for 15 minutes each at 42ºC.

scan_protocol: Slides were scanned for Cy3 and Cy5 fluorescence using Scanarray 5000 XL (GSI Lumonics) and quantified using the GenePix Pro 4.0 software (Axon Instruments Inc.)

data_processing: GenePix files were uploaded into the DNMAD module of the Gene Expression Profile Analysis Suite v3.1 (GEPAS; http://www.gepas.org) (Montaner et al. Next station in microarray data analysis: GEPAS. Nucleic Acids Research, 2006, 34 (Web Server issue): W486-W491.) and normalized using print tip loess without background substraction and flagged points were returned as missing values.

label_protocol: Fluorescent cDNA probe was generated from 2.5 micrograms of amplified RNA in a reaction volume of 25 microlitres containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 0.01 M DTT, 0.5 mM each dATP, dCTP and dGTP and 0.1 mM dTTP, 1.5 micrograms random hexamer primers, 0.6 mM FluoroLink Cy5-dUTP (Amersham) and 10 units of Superscript II reverse transcriptase (Invitrogen) incubated at 37ºC for 120 minutes. After RNA hydrolysis, 10 micrograms of Cot1 Human DNA (GIBCO) were added and the fluorescent probe was concentrated using Microcon YM-30 (Millipore)

extract_protocol: Total RNA was isolated using TRI-Reagent (Molecular Research Center, Inc.) followed by DNAse treatment using RNase-Free DNase (QIAGEN). Purity and quality of the samples was analyzed by agarose gel electrophoresis. cDNA was generated using 5 micrograms total RNA using SuperScript™ Choice System for cDNA Synthesis. Double-stranded cDNA was purified by phenol/chlorophorm followed by ethanol precipitation and subsequently used as template for T7 RNA polymerase amplification using the MEGAscript® T7 kit (Ambion). After DNase treatment, the amplified RNA was purified using RNeasy columns (QIAGEN), quantified spectrophometrically at 260 nm and analyzed by agarose gel electrophoresis.

tag: prostate_cancer_16 Gleason_score:3+4

label_protocol: Fluorescent cDNA probe was generated from 2.5 micrograms of amplified RNA in a reaction volume of 25 microlitres containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 0.01 M DTT, 0.5 mM each dATP, dCTP and dGTP and 0.1 mM dTTP, 1.5 micrograms random hexamer primers, 0.6 mM FluoroLink Cy3-dUTP (Amersham) and 10 units of Superscript II reverse transcriptase (Invitrogen) incubated at 37ºC for 120 minutes. After RNA hydrolysis, 10 micrograms of Cot1 Human DNA (GIBCO) were added and the fluorescent probe was concentrated using Microcon YM-30 (Millipore)


title: Cortical vs Spinal Cord NS (14136)

description: as per agilent protocol


title: prostate_cancer_10

tag: prostate_cancer_10 Gleason_score:3+2


title: prostate_cancer_17

tag: prostate_cancer_17 Gleason_score:3+3


title: prostate_cancer_14

tag: prostate_cancer_14 Gleason_score:3+4


title: prostate_cancer_13

tag: prostate_cancer_13 Gleason_score:2+4


title: prostate_cancer_15

tag: prostate_cancer_15 Gleason_score:3+4


title: prostate_cancer_12

tag: prostate_cancer_12 Gleason_score:3+3


title: prostate_cancer_11

tag: prostate_cancer_11 Gleason_score:4+3


title: prostate_cancer_07

tag: prostate_cancer_07 Gleason_score:4+5


title: prostate_cancer_04

tag: prostate_cancer_04 Gleason_score:3+4


title: prostate_cancer_09


title: prostate_cancer_05

tag: prostate_cancer_05 Gleason_score:3+4


title: prostate_cancer_08

tag: prostate_cancer_08 Gleason_score:4+3


title: prostate_cancer_01

description: Human prostate cancer hybridized against normal adjacent prostate tissue from the same patient.

hyb_protocol: Concentrated fluorescent cDNA probes (Cy3 and Cy5 labelled) were combined, ethanol precipitated and resuspended in 40 microlitres of SlideHyb Buffer #1 (Ambion). Ten micrograms of polyA (SIGMA) and 4 micrograms of yeast tRNA (GIBCO) were added as blocking agents and the samples were denatured at 95ºC for 2-3 minutes and applied to the prehybridized microarray slide. Prehybridization of microarray slides was performed in 4x SSC, 0.1% SDS and 0.002% BSA for 45 minutes at 65ºC. Hibridization was carried out at 42ºC for 15-20 hours. After hibridization, slides were washed 3 times in 2X SSC, 0.5 % SDS for 15 minutes each at 42ºC and then 3 times in 0.5X SSC, 0.5 % SDS for 15 minutes each at 42ºC.


title: prostate_cancer_02


title: CTB-3

description: Affymetrix

tag: First trimester placental villous material was digested with a 0.125% (v/v) trypsin solution (GIBCO, Life Technologies, Paisley, UK). The released cells were loaded on top of a Percoll (Pharmacia, Uppsala, Sweden) gradient ranging from 70 to 10% (v/v). After centrifugation, the band containing the trophoblasts was collected and sequentially purified using immunomagnetic beads (Dynabeads M-450, Dynal) conjugated to CD45 RB leukocyte common antigen (Dako, CA) and to fibroblast specific antibody A 502 (Dianova, Hamburg).

tag: Trophoblasts were cultured for 24 h in DMEM (GIBCO)


title: prostate_cancer_06

tag: prostate_cancer_06 Gleason_score:2+4


title: Layer_8w_rep2

status: Public on Jan 01 2009

city: Wilmington

first: Qi

last: Zheng

state: DE

address: 1701 Rockland Road

institute: Nemours Children's Health

description: 5 randomly selected male individuals were used to make the total RNA pool.

hyb_protocol: Affymetrix Gene Chip array hybridization was carried out according to Affymetrix Expression Analysis Technical Manual by GeneTech Biotechnology Limited Company (Shanghai, China). Following labeled cRNA fragmentation, 15 μg of cRNA were hybridized for 16 hr at 45℃ on Affymetrix Chicken Genome Array. Washing and staining were followed standard Affymetrix protocol.

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

data_processing: Raw intensity was processed by R packages. Background adjustment, probe-level normalization, pm-correction and probeset summary were achieved by RMA method in Affymetrix package. P/M/A calls were achieved by MAS5 method in Affymetrix package.

organism: Gallus gallus

source_name: Pectoralis major muscle, Layer, 8w

label_protocol: Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 2 ug total RNA.

growth_protocol: All chickens were kept in floor pens under the same temperature and lighting conditions. For the first two days after hatch, chicks were kept in floor pens at 35°C under constant light. From the post-hatching third day to 8 weeks, temperature was reduced from 35°C to 18°C in a stepwise manner and lighting schedule was maintained at a 18 h light and 6 h darkness cycle.

extract_protocol: Pectoralis major muscles were excised and immediately frozen in liquid nitrogen and then stored at -70 °C until RNA extraction. Trizol extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: Both two chicken strains were fed with commercial layer diet. All the chickens were weighed weekly and 5 individuals randomly selected from each group were sacrificed for tissue sampling.

tag: Breed

value: White Leghorn, Gender:male, Tissule:Pectoralis major muscle


title: Layer_6w_rep3

source_name: Pectoralis major muscle, Layer, 6w


title: Layer_8w_rep3


title: Layer_8w_rep1


title: Layer_6w_rep2


title: Layer_4w_rep2

source_name: Pectoralis major muscle, Layer, 4w


title: Layer_2w_rep3

source_name: Pectoralis major muscle, Layer, 2w


title: Layer_4w_rep3


title: Layer_4w_rep1


title: Layer_1d_rep2

source_name: Pectoralis major muscle, Layer, 1d


title: Layer_2w_rep2


title: Layer_6w_rep1


title: Layer_2w_rep1


title: Layer_1d_rep1


title: Broiler_8w_rep2

source_name: Pectoralis major muscle, broiler, 8w

value: AA, Gender:male, Tissule:Pectoralis major muscle


title: Broiler_6w_rep3

source_name: Pectoralis major muscle, broiler, 6w


title: Broiler_8w_rep1


title: Broiler_6w_rep2


title: Broiler_8w_rep3


title: Broiler_2w_rep3

source_name: Pectoralis major muscle, broiler, 2w


title: Layer_1d_rep3


title: Broiler_4w_rep3

source_name: Pectoralis major muscle, broiler, 4w


title: Broiler_1d_rep3

source_name: Pectoralis major muscle, broiler, 1d


title: Broiler_4w_rep1


title: Broiler_6w_rep1


title: Broiler_4w_rep2


title: Broiler_2w_rep1


title: 1 nM R1881, 10 nM  LBH589, rep 1

city: New York

first: Yu

state: NY

address: 1275 York Ave, Box 20

department: Human Oncology and Pathogenesis Program

institute: Memorial Sloan Kettering Cancer Center

description: 1 nM R1881, 10 nM  LBH589, rep 1

hyb_protocol: According to standard Affymetrix protocol.

data_processing: Data was analyzed using GeneSpring. Gene with intensity >250 either unstimulated or stimulated with R1881 was included for analysis. Data was normaled by medium of the chip. No per gene normalization was performed.

source_name: LNCaP Cells

growth_protocol: LNCaP cells were grown in charcoal stripped medium

extract_protocol: RNAeasy Kit

treatment_protocol: As per title for 16 hours, all in the presence of cyclohexamide


title: Broiler_2w_rep2


title: 1 nM R1881, 10 nM  LBH589, rep 2

description: 1 nM R1881, 10 nM  LBH589, rep 2

scan_protocol: According to standard Affymetrix protocol.


title: 1 nM R1881, 100 nM  LBH589, rep 1

description: 1 nM R1881, 100 nM  LBH589, rep 1

label_protocol: According to standard Affymetrix protocol.


title: 1 nM R1881, 100 nM  LBH589, rep 2

description: 1 nM R1881, 100 nM  LBH589, rep 2


title: 1 nM R1881, 0.5 µM SAHA, rep 1

description: 1 nM R1881, 0.5 µM SAHA, rep 1

tag: 1 nM R1881, 0.5 µM SAHA, rep 1


title: 1 nM R1881, 5 µM SAHA, rep 2

description: 1 nM R1881, 5 µM SAHA, rep 2

tag: 1 nM R1881, 5 µM SAHA, rep 2


title: Broiler_1d_rep1


title: Broiler_1d_rep2


title: 1 nM R1881, No HDACi, rep 1

description: 1 nM R1881, No HDACi, rep 1

tag: 1 nM R1881, No HDACi, rep 1


title: 1 nM R1881, 0.5 µM SAHA, rep 2

description: 1 nM R1881, 0.5 µM SAHA, rep 2

tag: 1 nM R1881, 0.5 µM SAHA, rep 2


title: 1 nM R1881, 5 µM SAHA, rep 1

description: 1 nM R1881, 5 µM SAHA, rep 1

tag: 1 nM R1881, 5 µM SAHA, rep 1


title: 1 nM R1881, 0.1 µM TSA

description: 1 nM R1881, 0.1 µM TSA


title: 1 nM R1881, 1 µM TSA

description: 1 nM R1881, 1 µM TSA

tag: 1 nM R1881, 1 µM TSA


title: Glis2 KO vs. Glis2 WT

city: RTP

first: NIEHS

last: Microarray Core

state: NC

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

description: Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol.  Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturer's protocol.    For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol.  Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol.    Chips were scanned with an Agilent Scanner and processed with the Agilent Feature Extraction Software (v8.1).

data_processing: Agilent Feature Extraction protocol, log2ratio Treated/Control

source_name: Glis2 KO kidney

label_protocol: Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol.  Starting with 0.5µg of total RNA, Cy3 labeled cRNA was produced according to manufacturer's protocol.

extract_protocol: Total RNA was collected using Trireagent (Sigma) following the manufacture's protocol.

tag: Kidneys were collected from 2 month-old Glis2 knockout mice.

source_name: Glis2 WT kidney

label_protocol: Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol.  Starting with 0.5µg of total RNA, Cy5 labeled cRNA was produced according to manufacturer's protocol.

tag: Kidneys were collected from 2 month-old Glis2 wild type mice.


title: No R1881, 10 nM  LBH589, rep 2

description: No R1881, 10 nM  LBH589, rep 2


title: No R1881, 100 nM  LBH589, rep 2

description: No R1881, 100 nM  LBH589, rep 2


title: No R1881, 10 nM  LBH589, rep 1

description: No R1881, 10 nM  LBH589, rep 1


title: No R1881, 5 µM SAHA, rep 2

description: No R1881, 5 µM SAHA, rep 2

tag: No R1881, 5 µM SAHA, rep 2


title: No R1881, 0.5 µM SAHA, rep 2

description: No R1881, 0.5 µM SAHA, rep 2

tag: No R1881, 0.5 µM SAHA, rep 2


title: No R1881, 1 µM TSA

description: No R1881, 1 µM TSA

tag: No R1881, 1 µM TSA


title: No R1881, 0.5 µM SAHA, rep 1

description: No R1881, 0.5 µM SAHA, rep 1

tag: No R1881, 0.5 µM SAHA, rep 1


title: No R1881, No HDACi, rep 2

description: No R1881, No HDACi, rep 2

tag: No R1881, No HDACi, rep 2


title: No R1881, 100 nM  LBH589, rep 1

description: No R1881, 100 nM  LBH589, rep 1


title: FL cells exposed to 0.2μM MNNG, biological rep2 (HG-U133A)

city: Hangzhou

first: Hongjuan

last: Li

state: Zhejiang

address: 388 Yuhangtang Road

country: China

institute: Zhejiang University School of Medicine

description: Gene expression data from FL cells exposed to 0.2μM MNNG

hyb_protocol: The biotinlyated cRNAs were fragmented and hybridized to the microarrays at 45°C for 16 h in the Hybridization Oven 640 rotating at 60 rpm. Following hybridization, the microarrays were washed and stained with streptavidin-phycoerythrin in the Fluidics Station 400,

scan_protocol: Based on the Affymetrix Microarray Suite 5.0 platform, GeneChips were scanned using the GeneChip Scanner 3000, and original Affymetrix .dat proprietary output files were generated.

data_processing: Affymetrix MAS5.0 was used to obtain gene expression signals and normalization of our arrays’ signals by global scaling to the target of 500 for U133A and U133B for each array. Detection call and change call were generated through single array analysis and two-array comparison analysis, respectively. A probe set was delimited as differentially expressed only if it showed at least one “Present” detection call and exhibited the same “Increase” or “Decrease” change calls more than three of four-time comparisons in the orthogonal comparisons of the two MNNG and two control treatment microarrays.

source_name: human amnion epithelial FL cells exposed to 0.2μM MNNG for 2 h, and subsequently recovered for additional 4 h

label_protocol: Standard Affymetrix Protocols (Affymetrix GeneChip Expression Analysis Technical Manual, rev.3. 2001)

growth_protocol: Human amnion epithelial FL cells were grown as monolayers in minimal essential medium (MEM, Gibco-Invitrogen, Carlsbad, CA) supplemented with 10% newborn bovine serum (Gibco-Invitrogen), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37°C.

extract_protocol: Total RNA was isolated with Trizol reagents (Invitrogen), and further purified using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturers’ protocols. Isolated total RNA was analyzed on an Agilent Bioanalyzer 2100 to verify that the starting material was of good quality.

treatment_protocol: At the logarithmic growth phase, the cultures were exposed to vehicle control (0.1%, V/V) and increasing doses (0.2, 1.0, 10.0μM) of MNNG for 2 h in a culture medium that did not contain 10% newborn bovine serum, followed by additional growth for 4 h in a fresh medium containing 10% newborn bovine serum. The cultures were rinsed twice with Hank’s buffer before each change of the medium.

tag: Strain

value: human amnion epithelial FL cells, Gender:female


title: FL cells exposed to 1.0μM MNNG, biological rep1 (HG-U133A)

description: Gene expression data from FL cells exposed to 1.0μM MNNG

source_name: human amnion epithelial FL cells exposed to 1.0μM MNNG for 2 h, and subsequently recovered for additional 4 h


title: No R1881, No HDACi, rep 1

description: No R1881, No HDACi, rep 1

tag: No R1881, No HDACi, rep 1


title: No R1881, 5 µM SAHA, rep 1

description: No R1881, 5 µM SAHA, rep 1

tag: No R1881, 5 µM SAHA, rep 1


title: FL cells exposed to 10.0μM MNNG, biological rep1 (HG-U133A)

description: Gene expression data from FL cells exposed to 10.0μM MNNG

source_name: human amnion epithelial FL cells exposed to 10.0μM MNNG for 2 h, and subsequently recovered for additional 4 h


title: Glis2 WT vs. Glis2 KO

hyb_protocol: For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol.  Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol.

scan_protocol: Chips were scanned with an Agilent Scanner and processed with the Agilent Feature Extraction Software (v8.1).


title: FL cells exposed to 10.0μM MNNG, biological rep2 (HG-U133A)


title: FL cells exposed to 1.0μM MNNG, biological rep2 (HG-U133A)


title: FL cells exposed to 0.2μM MNNG, biological rep1 (HG-U133A)


title: No R1881, 0.1 µM TSA

description: No R1881, 0.1 µM TSA

tag: No R1881, 0.1 µM TSA


title: FL cells exposed to DMSO vehicle control, biological rep2 (HG-U133A)

description: Gene expression data from FL cells exposed to DMSO vehicle control

source_name: human amnion epithelial FL cells exposed to DMSO vehicle control for 2 h, and subsequently recovered for additional 4 h


title: FL cells exposed to DMSO vehicle control, biological rep1 (HG-U133A)


title: FL cells exposed to 10.0μM MNNG, biological rep1 (HG-U133B)


title: FL cells exposed to 0.2μM MNNG, biological rep2 (HG-U133B)


title: FL cells exposed to 1.0μM MNNG, biological rep1 (HG-U133B)


title: FL cells exposed to 0.2μM MNNG, biological rep1 (HG-U133B)


title: FL cells exposed to DMSO vehicle control, biological rep2 (HG-U133B)


title: FL cells exposed to DMSO vehicle control, biological rep1 (HG-U133B)


title: Oral62

status: Public on Dec 31 2008

city: Vancouver

first: Ivy

last: Tsui

state: BC

address: 675 West 10th Avenue

department: Cancer Genetics and Developmental Biology

country: Canada

institute: BC Cancer Research Centre

hyb_protocol: Probes were combined, denatured and annealed in a solution containing 100 ug human Cot-1 DNA (Invitrogen) and 48 ul DIG Easy hybridization solution (Roche) containing sheared herring sperm DNA (Sigma-Aldrich) and yeast tRNA (Calbiochem). Probe was denautred at 85°C for 5 minutes, and repetitive sequences were blocked at 45°C for 1 hour prior to hybridization. The probe mixture was applied to the slide surface and hybridized for 36 hrs at 45°C. The arrays were washed 5 times in 0.1X SSC, 0.1% SDS at room temperature with agitation. Each array was then rinsed repeatedly in 0.1X SSC and dried by centrifugation.

scan_protocol: A charge-coupled device (CCD) scanner system (ARrayworx eAuto, API, Issaquah, WA) was used to determine the signal intensities of the Cyanine 5/Cyanine3 channels. Arrays were scanned at a 9.75um resolution; exposure times for each image wavelength were usually between 0.2 to 2 sec. Images were analyzed by SoftWoRx Tracker analysis software (Applied Precision, Issaquah, WA, USA).

data_processing: A three-step normalization procedure [Khojasteh et al. BMC Bioinformatics 6:274, 2005] was performed to each raw data.

label_protocol: Random prime probe labeling was performed to label sample and reference genomic DNA (250ng each). Each reaction was labeled with either  2 nmol of cyanine-3 or cyanine-5 dCTP.

extract_protocol: All lesoins were microdissected, and the dissected tissues were places in sodium dodecyl sulfate/proteinase K at 55°C and spiked twice a day for 72 hours with fresh proteinase K. Genomic DNA was extracted wth phenol/chloroform and precipictaed with ethanol.

tag: Formalin-fixed paraffin-embedded oral specimen

source_name: Normal male

tag: Novagen pooled normal male genomic DNA


title: Oral53


title: Oral69


title: Oral71


title: Oral70


title: Oral56


title: Oral94


title: Oral51


title: Oral47


title: Oral63


title: Oral60


title: Oral52


title: Oral49


title: Oral68


title: Oral67


title: Oral57


title: Oral43


title: Oral50


title: Oral58


title: Oral42


title: Oral44


title: Oral45


title: Oral55


title: Oral46


title: Oral41


title: Oral39


title: 1 nM R1881, No HDACi, rep 2

description: 1 nM R1881, No HDACi, rep 2

tag: 1 nM R1881, No HDACi, rep 2


title: Oral40


title: Oral31


title: Oral48


title: Oral32


title: Oral25


title: Oral24


title: Oral27


title: Oral29


title: Oral20


title: Oral18


title: Oral17


title: Oral22


title: Oral15


title: Oral13


title: Oral8


title: FL cells exposed to 1.0μM MNNG, biological rep2 (HG-U133B)


title: Oral28


title: Oral16


title: Oral12


title: Oral6


title: Oral36


title: Oral1


title: W3R vs Hy1R

city: Ede

first: Arno

last: Wegkamp

address: Kernhemseweg 2

country: Netherlands

institute: NIZO food research

description:
In these experiments we compare the gene expression of the Lactobacillus plantarum WCFS1 harbouring the control plasmid pNZ7021. This plasmid is a high copy vector and contains a chloramphenicol marker, a multiple cloning site (to insert genes), prior to the multiple cloning site the pepN promoter is located, this promoter is a strong promoter of the lactic acid bacterium Lactoccus lactis. The strain to which the gene expression is compared is Lactobacillus plantarum WCFS1 harboring plasmid pNZ7026, this plasmid contains the complete folate gene cluster of L. plantarum WCFS1. The folate genes on these plasmid have the following names while present on the chromosome of L. plantarum WCFS1, Lp_3294, Lp_3294, Lp_3295, Lp_3296, Lp_3297, Lp_3298 and Lp_3299. In short, we compare the gene expression of the control strain of L. plantarum with a L. plantarum strain that can produce high amounts of folate. Both strains were cultivated in quadruple on chemically defined medium as described by Teusink et al AEM 2005, we have used in this experiment a glucose concentration of 25mM and the total amount of para-aminobenzoicacid that used is 10mg/l. The type of cultivation we used was chemostat, after the strains were grown in batch the medium-in and medium-out pumps were turned on and thereby a growth rate of 0.35 h-1 was used. In steady state we have harvested RNA and the RNA was handled as described in this protocol.
As stated above, the control and folate overproducing L. plantarum strains were cultivated 4 independent times in chemostat experiments. The names that were given to the RNA from the control experiment were W2, W3L, W3R and Warno; alternatively, the names that were give to the RNA from the folate overproducing strain were called Hy2 (named after hyper), Hy1L, Hy1R and Hyparno.
We have used the following hybridisation scheme to compare the samples with each other.
W2 and Hy2, Hy2 and W3L, W3L and Hy1L, Hy1L and W3R, W3R and Hy1R, Hy1R and W2, W2 and W3R, Hy1L and Hy2, Warno and Hyparno, W3R and Warno, Hyparno and Hy2, Hyparno and W3L.

hyb_protocol: Application This protocol can be used  for hybridisation of  Cy3/Cy5 labelled cDNA to  11K Agilent slides. It is an  abbreviated form of, and  an addition to the  manufacturer's protocol  (also available as pdf in  BASE under "protocols" as  "Agilent hyb. protocol v  4.1"). All chemicals  mentioned in the  hybridisation kit are  supplied in the Agilent in  situ hybridisation kit-plus  (Order# 5184-3568, lab  Arno, room temperature).  Micro array slides,  hybridisation chambers and  gasket slides (Order#  G2534-60002) are  available in Arno's lab. 20x  SSC (see NIZO QA  T/BFC063) is needed in  the wash solutions and has  to be made prior to the  washing steps. Hybridisation Tips When the  volume of the required  amount of labelled cDNA  exceeds 40 µl evaporate  the excess of liquid in the  SpeedVac (reduction from  120 to 40 µl takes about  45 min without heating) Be very sure that  you make 210 µl of  hybridization mix, otherwise  the array chamber will not  be filled to at least 50%.  This will cause a "black  hole" in the centre of the  array image. In case you  conclude that you do not  have enough hybridization  mix you can add some  hybridization buffer (mix of  equal volumes water and  2x hybridization buffer). Once the  chamber is closed, mark  the level of buffer on the  gasket slide (the one with  the rubber rings) using a  fine-liner. This allows you  to check afterwards  whether leakage has  taken place. Cy-dye  bleaching has been shown  to be promoted by light,  ozone and humidity  (relevant for dried slides).  On hot summer days,  humid days or in the  vicinity of UV lamps dyes  may bleach very quickly. Hybridisation Special powder free gloves  are available for working  with slides. Avoid too much  light during the procedure  to prevent the cyanine  dyes from bleaching. 1.	Take the  necessary number of  Agilent slides from the  desiccator and close the  desiccator properly. Notate  the serial numbers in your  journal. The side containing the  bar code and "Agilent"  label contains the spots,  the side with the bar code  and the slide number label  is the back. The array that  is furthest away from the  label is array number 1, the  closest is array number 2  (see Figure 1). 2.	Prepare 0.3  g of  Cy3 and 0.3  g of Cy5  labelled cDNA's as  described in "cDNA  synthesis and labelling"  each in a volume of 40 µl  (see tips if concentration is  too low, or add water if  concentration is too high.  Also see note below) 3.	Mix the Cy3 and  Cy5 labelled cDNA's in an  amber Eppendorf tube 4.	Heat at 98 °C for  3 minutes and cool down  to room temperature (use  clamp or pierce a hole in  the lid to avoid  spontaneous opening of  the lids when heating) 5.	Add 25 µl of 10x  control (freezer, Arno) 6.	Add 105 µl 2x  hybridisation buffer  (hybridisation kit) The total volume will now  be 210 µl 7.	Mix (no vortex to  avoid bubble formation)  and spin briefly to collect  all of the mix at the bottom  of the tube 8.	Put a gasket  slide (G2534-60002) in the  microarray hybridisation  chamber (G2534A) and  pipet the hybridisation  mixtures in the centre of  the array frames without  introduction of bubbles 9.	Put the  microarray slide slightly on  top of it without  introduction of bubbles (the  side containing the bar  code and "Agilent" label  has to face towards the  hybridation mixture). The  easiest way to achieve this  is to put one short side of  the microarray slide on the  gasket slide and slowly  lower the array slide 10.	Place the upper  part of the hybridisation  chamber (chamber cover)  and the chamber clamp  and close this by turning  the thumbscrew clockwise  and tighten it firmly by hand  (see Figure 2) Do not use tools to tighten  the thumbscrew when  closing the hybridisation  chamber!! 11.	Take the  hybridisation chamber and  turn it vertically to check  for correct bubble  formation. Bubbles are  correct when they move  freely in the hybridisation  chamber and the mixture  can reach the complete  surface of the slide upon  turning of the slide. Also  check whether the  chamber is filled to at least  60% of its total volume 12.	Hybridise for 17  hours at 60 °C. If  necessary balance the  rotor with empty  hybridization chambers Note about the amount of  cDNA to be used per array We are still discussing  whether the amount of  cDNA to be used on a  microarray should be  normalized with respect to  the concentration of cDNA  measured in the sample or  with respect to the  concentration of Cy-dye  measured. Amersham  recommends the second  option. However, until now  we have used the first  option (0.3  g cDNA per  array). Until now we have  observed ratios of  Cy-dye/cDNA of 0.03 to  0.12 pmol/ng yielding good  final results.   Washing Tips Before starting  the washing steps prepare  all washing solutions and  put them in washing trays  (lab Arno) If more than one  slide has to be washed,  separate all array and  gasket slides as described  and keep the array slides  submerged in solution 1  until all slide sandwiches  are separated. Continue  with the washing steps for  all slides at the same time. To avoid  exposure to light place a  box over the washing trays  when washing the slides. Preparation of wash  solutions Wash solution 1 (6x SSC,  0.005% Triton X-102) 700 ml	milliQ 300 ml	20x SSC  (NIZO-SOP T/BFC063) 0.500 ml	10% Triton  X-102 (hybridization kit) Mix the  components and pass the  solution through a 0.22 µm  filter Store at room  temperature  Wash solution 2 (0.1x  SSC, 0.005% Triton X-102) 995 ml	milliQ 5.0 ml	20x SSC  (NIZO-SOP T/BFC063) 0.500 ml	10% Triton  X-102 (hybridization kit) Mix the  components and pass the  solution through a 0.22 µm  filter Store at 4 ºC Washing 1.	After  hybridisation remove the  glass slide from the  hybridisation chamber and  separate the glass slides  completely submerged in  wash tray 1 containing  wash solution 1 at room  temperature by inserting  the blunt tip of the plastic  tweezers at the bar code  end of the slide sandwich  and gently turning the tip of  the tweezers. 2.	Transfer the  slides to wash tray 2  containing wash solution 1  and a magnetic stirrer and  place this on a magnetic  stir plate. Wash the slides  for 10 minutes at room  temperature at medium  stirring. 3.	Transfer the  slides to wash tray 3  containing pre-cooled  wash solution 2 and a  magnetic stirrer and place  it in another tray filled with  melting ice on a magnetic  stir plate. Wash for 5  minutes at medium stirring. 4.	Dry the slides by  blowing all droplets of  washing solution to the  edges of the slides using a  nitrogen airgun (Arno).  Adjust the manometer to 1  atm. 5.	Put the slides in  a dark dust-free box  (microscope slide box, at  the reader) and scan the  slide immediately (see  protocol "Scanning of  DNA microarrays").    Figure 1. Our convention  for numbering the arrays on  an Agilent slide.   Figure 2. Turning the  thumbscrew hand-tight.

scan_protocol: Application This procedure can be  followed to scan DNA  micro array slides on the  Scan Array Express  microarray scanner. Operating the array  scanner 1.	Turn on the  scanner and the computer  (in this order) 2.	Login: scanner,  password: scanner2 3.	double click the  'ScanArray Express' icon 4.	Switch on lasers  1 and 3 at least 15 minutes  prior to scanning arrays 5.	Put focus on file This is to prevent you from  accidentally turning of the  laser again after the 15  minutes warming-up phase 6.	Press 'Scan |  Prescan' 7.	Put resolution at  50 nm, and PMT value for  both channels at for  instance 70% (spotted  arrays) or 40% (Agilent  arrays) 8.	Press 'Start' 9.	Press 'Palette,'  'Green' as soon as  projection of the image has  started (first dye), select  the red colour as soon as  scanning of the second  layer (second dye) has  started 10.	Select frame for  high resolution scan, adjust  resolution (to 10 nm) and  scan the array for both  dyes. Adjust PMT values  to balance signals  obtained for both channels Balance both  channels as well as  possible to achieve a  comparable average  intensity for both channels.  This avoids large  normalisation corrections  during analysis of the data,  so decreases the chance  on introduction of  artefacts. Make at least  one scan in which none of  the spots are saturated.  Run this scan at for  instance PMT = 40% and  step-wise increase the  PMT level until both  channels have comparable  intensities (e.g. 1000  intensity units) 11.	Press 'File |  Save' to save the files,  'Save all' saves both dye  layers Create a new  folder for each array 'Save all' saves  the signals from both layers  in individual files It's possible to  save the 'composite' file in  .jpg format, however this is  not necessary since al  data are in the original files  as well 12.	Switch off the  lasers and the scanner  after use Tips Allow the lasers  to warm for at least 15  minutes   Quantification of spots 1.	Start Imagene by  double clicking the  'Imagene' icon 2.	Load tiff files of  both channels 3.	Load the  template file from the folder  'GAL files' located on the  desktop Every batch of arrays has  it's own GAL file. The  name of the GAL file  corresponding to the batch  of slides can be found  under 'Array LIMS | Array  designs' as 'print  map/reporter map' in BASE  (http://kluyver/base). (e.g.  LpGn07.gal for the several  batches of spotted L.  plantarum arrays from  Groningen and  '012731_D_20040830.gal'  that was edited to  lpag01.gal for the 1st  design of Agilent L.  plantarum slides) 4.	Adjust frame by  pressing the 'Rectangle  adjust' button, drag the  circles on the spots,  starting with the left-up  spot (this is not necessary  for Agilent slides since they  are recognised  automatically) 5.	Press the  'Automatically place grid'  button and check by eye if  the grid was placed  correctly 6.	Press 'Quantify'  (a table with spot id's and  intensities is produced) 7.	Save files (for  both dyes) in the same  directory as the original tiff  files are located just by  selecting (single click) the  directory (don't open!) and  press the 'Save' button 8.	Remove slides  by pressing 'File,' 'Remove  selected images' for both  channels before loading  and quantification of other  arrays (only necessary  when more slides are to be  quantified) Repeat steps 14, 16-20 to  load and quantify multiple  slides after the current  slides have been removed.  Repeat step 15 when  another type/batch of  slides is used. 9.	Import data in  BASE  (http://kluyver/base)   Although the choice is  completely arbitrary, for  clarity always use channel  1 for the Cy3 label and  channel 2 for the Cy5 label  to avoid accidental  swapping of labels during  data analysis.

data_processing: After blank spots had been  removed, array  measurements were  normalized by local fitting  of an M-A plot applying the  loess algorithm, using the  Limma package in R  (http://www.r-project.org).  Spot intensities in both  channels (I1 and I2) were  thus converted to M-A  coordinates, where M =  log2(I1/I2) and A =  log2(I1/I2)/2. Normalized  intensities were used for  further analysis.   The statistical significance  of differences was  calculated from variation in  biological replicated, using  the eBayes function in  Limma (cross-probe  variance estimation) and  Holmes determination of  significance. Only genes  with a log2 ratio of -1 and  +1 and a Holmes value  less than 0.1 were  considered significant.

organism: Lactiplantibacillus plantarum

source_name: Lactobacillus plantarum pNZ7021 (2) = W3R

label_protocol: General comments Tips Be sure that  your RNA is of good  quality before you start. Use PCR  machine 3 or 4 (suitable  for 0.2 ml tubes) for  incubation steps,  (program 70marc and  42marc). Place all the  reagents (except for the  RT enzyme) on ice to  thaw. Chemicals  descriptions can be  found on page 6. All reagents  are in room A128. If you  take a new batch,  please inform beheerder  (= Marc Stevens). This protocol  is made for the so-called  "Home-made kit". The  protocol for the  Amersham post-labelling  kit is the same, except  for the chemical's name.  Please follow the  manufacturer's protocol  when in doubt. A micro-array  file can be found in  room A128 with more  information, order  numbers and  manufacturer's  protocols. While  purifying the labelled  cDNA, the column  should still be coloured  even after tree wash  steps, when you have  labelled total RNA from  bacteria. If you do not  see the dye colours at  this stage, you will  probably not have  enough labelled cDNA  to obtain good  hybridization results. Labelled  cDNA can be stored at  -20°C, but only for a  short period. In  particular the Cy5 label  quickly bleaches. Do  not use labelled DNA  older than a few days,  or you may get very low  signal on the array. Cy-dye  bleaching has been  shown to be promoted  by light, ozone and  humidity. On hot summer  days, or in the vicinity of  UV lamps dyes may  bleach very quickly. Prepared  Cy-dyes can be stored  up to tree months at  -80°C. Note that the  relation between the  amount of RNA that  goes into the reaction  and the amount of  cDNA synthesized is not  proportional (see figure  below by Roger  Bongers)   Things that can go  wrong (and already  went wrong once) Wash with  EtOH (80%), elute with  NaCO3 during the first  purification. Work in the  dark from the moment  you see colours in your  sample. Add 40 ml of  100% EtOH to the wash  buffer of the GFX  column purification kit.   cDNA Synthesis General comments Nuclease-free  water is available in  PCR kits or can be  ordered from commercial  suppliers. It is not useful  to add more than 25 µg  RNA to the Annealing  Mix, because this will  not lead to higher cDNA  yields (see figure on  page 1). Annealing Annealing Mix x µl	RNA  preparation (25 µg RNA  for spotted arrays or 5 to  10 µg RNA for Agilent  arrays, see tips) y µl	nuclease-free  water 1 µl	Random  Nonamers 11 µl	TOTAL  1.	Mix Annealing  Mix in a 200 µl PCR  tube gently by pipetting  up and down 2.	Incubate for 5  min. 70°C 3.	Cool at room  temperature for 10 min.  (annealing) 4.	Spin down  the mixture to the  bottom of the tube 5.	Place on ice Reverse transcription Reverse Transcription  Mix 4  l	5x buffer 2  l	0.1 M DTT 2  l	dNTP-mix  (with AA-dUTP [home  labelling kit] or 1 + 1  l  for Amersham kit, see  Manufacturers Protocol) 1  l	TMIII (keep  only briefly outside   20°C) 20  l	TOTAL  1.	Mix by very  gently by pipetting up  and down (vigorous  pipetting will denature  the enzyme!) Before pipetting the  enzyme: spin down the  residue in the tube to be  sure that all enzyme is  finished before starting a  new kit! 2.	Incubate for 3  hrs at 42°C (in  PCR-machine) 3.	Cool on ice  for immediate  purification or place at   20°C for storage Degradation of mRNA 1.	Add 2  l 2,5 M  NaOH 2.	Mix (vortex)  and spin down 3.	Incubate for  15 min. at 37°C 4.	Add 10  l 2 M  HEPES free acid 5.	Mix (vortex)  and spin down 6.	Ready for  purification or store at   20°C   Labelling of cDNA with  cyanine dyes General comments Use 80%  ethanol as wash buffer. The small  amount of Tris present  in the kit wash buffer  can react with the  Cy-dye NHS Esters,  reducing the yield of  labelling during the  coupling step. Prepare fresh  Sodium Bicarbonate 0.1  M, pH 9.0 (see page 6)  immediately prior to use  in a labelling reaction, or  use an aliquot from a   20°C stock (do not  freeze it again!). Purification of amino  allyl-modified cDNA 1.	For every  cDNA reaction to be  purified, place one  CyScribe GFX column  into a clean collection  tube. Add 500  l of  capture buffer to each  CyScribe GFX column 2.	Transfer the  unpurified cDNA  products into each  CyScribe GFX column;  mix the cDNA by gently  pipetting up and down 5  times 3.	Centrifuge  each column in a micro  centrifuge at 13.800 x g  for 30 seconds 4.	Remove the  CyScibe GFX column  and discard the liquid at  the bottom of each  collection tube. Return  each CyScribe GFX  column into the used  collection tube 5.	Add 600  l of  80% ethanol to each  column and centrifuge  at 13.800 x g for 30  seconds 6.	Remove the  CyScibe GFX column  and discard the liquid at  the bottom of each  collection tube. Repeat  the wash step twice for  a total of three washes.  After the final wash,  discard the liquid and  place the column back  in the used collection  tube 7.	Centrifuge  each column in a  microcentrifuge at  13.800 x g for an  additional 10 seconds to  remove all wash buffer  in the tip of the column.  Discard the collection  tube 8.	Transfer each  CyScribe GFX column to  a fresh 1.5 ml micro  centrifuge tube and add  60 µl of fresh 0.1 M  sodium bicarbonate  pH9.0 directly to the top  of glass fiber matrix in  each CyScribe GFX  column. It is crucial that  the elution buffer  completely covers the  membrane 9.	Incubate the  CyScribe GFX column at  room temperature for 1-5  minutes. Centrifuge at  13.800 x g for 1 minute  to collect the purified  labelled cDNA 10.	Repeat this  elution step one more  time 11.	Proceed  immediately to the  labelling reaction Labelling of amino  allyl-modified cDNA with  Cy-dye 1.	Add the  amino allyl modified  cDNA (in 0.1 M sodium  bicarbonate) directly into  one aliquot of CyDye  NHS Ester. Resuspend  the ester completely by  pipetting several times  and transfer the solution  to an amber 1.5 ml tube 2.	Incubate at  room temperature, in the  dark for 60 to 90  minutes 3.	Add 15 µl 4 M  Hydroxylamine to each  coupling reaction 4.	Mix by stirring  and incubate at room  temperature, in the dark,  for 15 minutes 5.	Proceed  directly to purification of  Cy-dye labelled cDNA Purification of Cy-dye  labelled cDNA 1.	For every  cDNA reaction to be  purified, place one  CyScribe GFX column  into a clean collection  tube. Add 500 µl of  capture buffer to each  CyScribe GFX column 2.	Transfer the  unpurified cDNA  products into each  CyScribe GFX column;  mix the cDNA by gently  pipetting up and down 5  times 3.	Centrifuge  each column in a micro  centrifuge at 13.800 x g  for 30 seconds 4.	Remove the  CyScibe GFX column  and discard the liquid at  the bottom of each  collection tube. Return  each CyScribe GFX  column into the used  collection tube 5.	Add 600 µl of  wash buffer (from the  kit) to each column and  centrifuge at 13.800 x g  for 30 seconds 6.	Remove the  CyScibe GFX column  and discard the liquid at  the bottom of each  collection tube. Repeat  the wash step twice for  a total of three washes.  After the final wash,  discard the liquid and  place the column back  in the used collection  tube 7.	Centrifuge  each column in a  microcentrifuge at  13.800 x g for an  additional 10 seconds to  remove all wash buffer  in the tip of the column.  Discard the collection  tube 8.	Transfer each  CyScribe GFX column to  a fresh 1.5 ml amber  micro centrifuge tube  and add 60 µl of elution  buffer (from the kit)  directly to the top of the  glass fiber matrix in each  CyScribe GFX column. It  is crucial that the elution  buffer completely covers  the membrane CyDye-labelled probes  bind tightly to GFX resin.  Never use water to  elute these labelled  probes because probe  yield will be inconsistent  and lower than that  obtained with elution  buffer Pre-warming elution  buffer to 65°C might  increase the labelled  cDNA yield by  approximately 5% 9.	Incubate the  CyScribe GFX column at  room temperature for 1-5  minutes. Centrifuge at  13.800 x g for 1 minute  to collect the purified  labelled cDNA 10.	Repeat this  elution step one more  time 11.	Measure the  concentration of cDNA,  and the amount of  incorporated Cy-dye  using the "microarray"  program of the  nanodrop (see note  below). 12.	Ready for  hybridization or store at   20°C Notes about labelled  cDNA absorption  spectrum The 260/280  nm absorption ratio  should be in the range  1.6 to 1.8 The spectrum  of the incorporated  Cy-dye as recorded with  the nanodrop gives you  important information  about the fluorescence  processes going on. In  particular, a high ratio of  the first absorption peak  relative to the second  will indicate high  self-quenching due to  overloading of the  cDNA with dye. If the  peaks are almost  equally high then the  labelling density is too  high and fluorescence  will be low due to  self-quenching.   Home-made labelling kit  components TMIII =  Superscript TMIII  (Invitrogen, order #  18080-044) DTT is  supplied with TMIII  enzyme 5 x Buffer is  supplied with TMIII  enzyme GFX Columns  can be ordered from  Amersham Bioscience,  order # 27-9606-02 NaOH,  HEPES, Hydroxylamine,  and Sodium  Bicarbonate solutions  should be made by the  researcher. dNTP's can  be ordered from  Amersham Bioscience,  order # 27-2035-01 The random  nonamer stock (to be  diluted 1/20) can be  found in room A128  Hydroxylamine 4 M	 Hydroxylamine Prepare freshly  Sodium Bicarbonate 0.1  M, pH 9.0 0.84 g	NaHCO3 90 ml	Milli-Q water Adjust to  exactly pH 9.0 with 1 M  NaOH Adjust volume  to 100 ml with Milli-Q  water Store in  aliquots or prepare  freshly. Do not freeze  again after use  Random Nonamers 2 µl	random  nonamer stock (27.1  µg/µl) 38 µl 	nuclease-free  water  AA-dUTP 1 mg	AA-dUTP  (Sigma, order # A0410) 20 µl	K-phosphate,  0.1M, pH 7.5  dNTP-mix 5 µl	100 mM of  each dATP, dCTP and  dGTP 3 µl	100 mM  dTTP 2 µl	AA-dUTP-mix Add 40 µl	Water  Cy5-dye or Cy3-dye  NHS Ester 1 tube	Cy5 or Cy3  monofunctional reactive  dye (Amersham, order #  PA23001) 40 µl 	DMSO Store in 8 x 5 µl aliquots  at  80°C up to 3 months Use 5 µl per labelling   Toxic chemicals  HYDROXYLAMINE  (See Chemical safety  cards on  http://kluyver/intranet/) Chemical  dangers: May explode  on heating above 70°C  or when exposed to  open flame. The  substance decomposes  rapidly at room  temperature, especially  in presence of moisture  and carbon dioxide, and  violently on heating  producing toxic fumes  including nitrogen  oxides. The solution in  water is a weak base.  Reacts violently with  oxidants, metals such as  finely divided zinc, some  metal oxides,  copper(II)sulfate and  phosphorus chlorides  causing fire and  explosion hazard. Effects of  short-term exposure:  The substance irritates  the skin and the  respiratory tract, and is  corrosive to the eyes.  The substance may  cause effects on the  blood, resulting in  formation of  methaemoglobin. The  effects may be delayed.  Medical observation is  indicated. Effects of  long-term or repeated  exposure: Repeated or  prolonged contact may  cause skin sensitization.  The substance may  have effects on the  blood, resulting in  formation of  methaemoglobin and  the consequent  anaemia.

extract_protocol: Isolation of RNA General comments Both  Biophenol and Acidic  phenol (Sigma) are  suitable for application  in the Extraction  Mixture. The ratio of  pellet to Extraction  Mixture seems to affect  the RNA amounts and  quality. 50 ml MRS  culture of an OD600 of  1 gives reproducibly  good results. Five times  this amount, however,  yielded poor quality  RNA (Personal  communication from  Arno Wegkamp and  Mariela Hebben). The  quenching buffer  contains methanol.  Supernatant from the  quenching procedure  should be discarded in  the appropriate  container. The  extraction buffer  contains phenol. Phenol  is a toxic compound and  waste containing phenol  should be discarded in  the appropriate  containers. Collecting cells Quenching Buffer 66.7 mM	HEPES (pH  6.5) 60%	Methanol  Extraction Mixture 500 µl	 Phenol/Chloroform 30 µl	10% SDS 30 µl	3 M NaAc  (pH 5.2) 500 mg	glass-beads  (75-150 µm) 400 µl	TE buffer (or  MRS) Prepare in a screw-cap  tube  1.	For each  sample prepare a tube  with Extraction Mixture Make sure these  components are mixed  freshly each time, so do  not freeze tubes  containing Extraction  Mixture. 2.	Harvest cells  and quench them  immediately in  Quenching Buffer. Add  4 volumes of Quenching  Buffer ( 40°C) to 1  volume of cell culture 3.	Mix and store  the sample at -40°C 4.	Centrifuge  cells at  20 C at 9000  rpm for 10 minutes  (Centrifuge RC5B, room  A156) 5.	Discard the  supernatant; keep the  cell pellet cooled in  ethylene glycol (60 %)  and work quickly to  prevent condensation of  water. Transfer the  pellet with a pre-cooled  spatula to a screw-crap  tube with Extraction  Mixture The supernatant  contains methanol and  should be discarded in  the appropriate  container 6.	Mix the cell  pellet thoroughly with  Extraction Mixture by  shaking by hand 7.	Freeze the  tube immediately in  liquid nitrogen 8.	Store at  80°C  or isolate RNA  immediately RNA isolation Tips: Work as  quickly as possible and  keep the samples cool. Do not break  too much samples at  once (max 4) to enable  you to work quickly. Break the  cells immediately in  frozen condition. Do not  thaw the samples from   80°C.  Keep your  samples cool during  processing. Use filter tips  to avoid contamination  from pipets. Wear gloves  and lab coat. For more tips  look at the Ambion  website:  http://www.ambion.com The following steps  should be performed as  swiftly as possible  before cDNA labelling. 1.	Break cells in  the Savant FastPrep  FP120. Three times 40  seconds at speed 4.0  (cool cells between  steps if necessary) 2.	Centrifuge for  1 minute at 4°C in an  Eppendorf centrifuge at  20.800 g. 3.	Transfer 500  µl of the supernatant to  a new tube. Add 400 µl  chloroform (chloroform  should be cooled at  4°C) 4.	Centrifuge for  1 minute in an  Eppendorf centrifuge at  full speed (4°C). 5.	Take the  supernatant and mix it in  1:1 ratio with the  capture buffer of the  RNA-isolation kit. 6.	Continue with  the "High Pure RNA  Isolation Kit" from  Roche (order # 1 828  665) 7.	Incubate for  60 minutes with DnaseI,  elute with 50 µl elution  buffer 8.	Store the  RNA in at least 2  aliquots, one of 20 µl  (for concentration and  quality analysis and  labelling) and one  back-up of 30 µl at   80°C. RNA quality control Measuring the RNA  concentration  Measure the  concentration of RNA at  260 nm by dissolving 1  µl of a preparation in  500 µl of water. 1 absorption  unit at 260 nm is  equivalent to 40 µg  RNA/ml. The  A260/A280 ratio should  be in the range of  1.9-2.0. The closer to  2.0, the more pure the  RNA will be. When the  ratio is lower than 1.9,  DNA is still present.  When lower than 1.8,  proteins are still present. The  recommended RNA  concentration for  labelling is 20-25 µg /µl . The minimal  RNA concentration  should be 1 µg/µl. Quality check on the  Bioanalyzer First  determine the  RNA-concentration  using the "nanodrop". The  Bioanalyzer can be  found in room A230. For the quality  check you have to use  the Nano-assay chips. Try to apply  12 samples to the  Bioanalyzer, but, on the  other hand, do not wait  for a week to analyse  your samples. Please follow  the instructions of the  manufacturer or ask the  instrument custodian (=  Anne Wiersma). The ratio  between the 23S and  the 16S RNA should be  at least 1.6. Lower ratios  are an indication of  extensive RNA  degradation. The  "zero-line" in the  electrogram should be  more or less straight  (when in doubt, ask an  experienced  researcher). The analysis  on the Bioanalyzer is a  qualitative assay, not a  quantitative assay. The  concentrations found in  the spectrophotometer  are more reliable.

source_name: Lactobacillus plantarum pNZ7026 (3) = Hy1R


title: Warno vs Hyparno

source_name: Lactobacillus plantarum pNZ7021 (4) = Warno

source_name: Lactobacillus plantarum pNZ7026 (4) = Hyparno


title: Hy1L vs W3R

source_name: Lactobacillus plantarum pNZ7026 (2) = Hy1L

tag: Lactobacillus plantarum pNZ7021 (2) = W3R


title: Hyparno vs hy2

tag: Lactobacillus plantarum pNZ7026 (4) = Hyparno

source_name: Lactobacillus plantarum pNZ7026 (1) = Hy2


title: Hyparno vs W3L

source_name: Lactobacillus plantarum pNZ7021 (3) = W3L

tag: Lactobacillus plantarum pNZ7021(3) = W3L


title: Oral89


title: W3R vs Warno

tag: Lactobacillus plantarum pNZ7021 (4) = Warno


title: Oral93


title: W3L vs Hy1L

tag: Lactobacillus plantarum pNZ7021 (3) = W3L

tag: Lactobacillus plantarum pNZ7026 (2) = Hy1L


title: W2 vs W3R

source_name: Lactobacillus plantarum pNZ7021 (1) = W2


title: Hy2 vs W3L

tag: Lactobacillus plantarum pNZ7026 (1) = Hy2


title: Oral92


title: Oral87


title: Hy1L vs Hy2


title: Oral88


title: Oral90


title: Oral91


title: Hy1R vs W2

tag: Lactobacillus plantarum pNZ7026 (3) = Hy1R

tag: Lactobacillus plantarum pNZ7021 (1) = W2


title: Oral81


title: Oral85


title: FL cells exposed to 10.0μM MNNG, biological rep2 (HG-U133B)


title: W2 vs Hy2


title: Oral84


title: Oral80


title: Oral79


title: Oral83


title: Oral78


title: Oral82


title: Oral86


title: Oral77


title: Oral76


title: Oral66


title: Oral65


title: Oral73


title: Oral38

source_name: Normal female

tag: Novagen pooled normal female genomic DNA


title: Oral19


title: Oral30


title: Oral34


title: Oral37


title: Oral14


title: Oral23


title: Oral72


title: Oral54


title: Oral74


title: Oral64


title: Oral61


title: Oral35


title: Oral59


title: Oral21


title: Oral26


title: Oral33


title: Oral10


title: Oral5


title: Oral7


title: Oral9


title: Oral11


title: Oral3


title: Oral4


title: rat liver treated with OLT, rep3

status: Public on Dec 30 2008

city: Memphis

first: Thomas

middle: R

last: Sutter

state: TN

address: 3774 Walker Ave.

department: Biology

institute: University of Memphis

description: Gene expression data from livers of rats that were treated with OLT

hyb_protocol: Following fragmentation, 20ug of cRNA were hybridized for 16 hr at 45C on GeneChip Rat Genome U34 Array Set RG-U34A. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

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

data_processing: The data were analyzed with Bioconductor using GC-RMA analysis.

source_name: rat liver treated with OLT

label_protocol: Enzo labeling kit

extract_protocol: Stat-60 extraction

treatment_protocol: Rats were fed with Harlan Teklad diet treated with 100?l of 4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione  (OLT) once every other day for 5 days.

tag: Male Fischer F344 rats (80-100g)


title: rat liver treated with OLT, rep2


title: rat liver treated with D3T, rep3

description: Gene expression data from livers of rats that were treated with D3T

source_name: rat liver treated with D3T

treatment_protocol: Rats were fed with Harlan Teklad diet treated with 100?l of 3H-1,2-dithiole-3-thione  (D3T) once every other day for 5 days.