title: HPV16 immortalized keratinocytes_60hs_2(rep1)

description:
HPV16 immortalizes keratinocytes_60hs in culture_2
reference: NHFK_3hs in culture_1
amplified RNA


title: HPV16 immortalized keratinocytes_3hs_2(rep1)

status: Public on Aug 25 2007

type: other

city: Sao Paulo

first: Lara

last: Termini

state: SP

address: Rua Joao Juliao, 245 - 1th floor

country: Brazil

institute: Ludwig Institute for Cancer Research

description:
HPV16 immortalizes keratinocytes_3hs in culture_2
reference: NHFK_3hs in culture_1
amplified RNA

hyb_protocol: Hibridization - Solution: 5 X SSC; 0,1% SDS; 4 ug poly A DNA; 4 ug Cot1 DNA; 5X Denhardt´s ; 10 ug Esperma de salmão,  Formamida 25%. / Temperature:14 a 24 hs / Time:42 C / Touch Down: Washing - Round 1 - Solution: 0,1 X SSC / Temperature:sol 42C / Lavagem TA / Time:5 min / Washing Number: 1 Washing - Round 2 - Solution: 0,1 X SSC / Temperature:sol 42C / Lavagem TA / Time:10 min / Washing Number: 2 Washing - Round 3 - Solution: 0,1 X SSC / Temperature:sol 42C / Lavagem TA / Time:2,5 min / Washing Number: 2

scan_protocol: Hardware: Perkinelmer / Software: ScanArray

data_processing: Lowesss

molecule: other

organism: Homo sapiens

tag: HPV16 immortalized keratinocytes

tag: normal human keratinocytes


title: HPV16 immortalized keratinocytes_60hs_2(rep2)


title: HPV16 immortalized keratinocytes_3hs_1(rep2)

description:
HPV16 immortalizes keratinocytes_3hs in culture_1
reference: NHFK_3hs in culture_1
amplified RNA


title: HPV16 immortalized keratinocytes_3hs_2(rep2)


title: HPV16 immortalized keratinocytes_3hs_1_TNF(rep1)

description:
HPV16 immortalizes keratinocytes_3hs TNF_1
reference: NHFK_3hs in culture_1
amplified RNA

tag: HPV16 immortalized keratinocytes TNF 3hs


title: HPV18 immortalized keratinocytes_60hs_TNF_1(rep2)

description:
HPV18 immortalizes keratinocytes_60hs _TNF_1
reference: NHFK_3hs in culture_1
amplified RNA


title: HPV16 immortalized keratinocytes_60hs_1(rep2)

description:
HPV16 immortalizes keratinocytes_60hs in culture_1
reference: NHFK_3hs in culture_1
amplified RNA


title: HPV18 immortalized keratinocytes_60hs_1_TNF(rep1)

tag: HPV18 immortalized keratinocytes _60hs_TNF


title: HPV18 immortalized keratinocytes_60hs_2_TNF(rep1)

description:
HPV18 immortalizes keratinocytes_60hs _TNF_2
reference: NHFK_3hs in culture_1
amplified RNA

tag: HPV18 immortalized keratinocytes 60hs TNF


title: HPV16 immortalized keratinocytes_3hs_1(rep1)


title: HPV18 immortalized keratinocytes_3hs_2_TNF(rep2)

description:
HPV18 immortalizes keratinocytes_3hs TNF_2
reference: NHFK_3hs in culture_1
amplified RNA

tag: HPV18 immortalized keratinocytes 3hs TNF


title: HPV18 immortalized keratinocytes_60hs_1(rep2)

description:
HPV18 immortalizes keratinocytes_60hs in culture_1
reference: NHFK_3hs in culture_1
amplified RNA

tag: HPV18 immortalized keratinocytes


title: HPV18 immortalized keratinocytes_60hs_2(rep2)

description:
HPV18 immortalizes keratinocytes_60hs in culture_2
reference: NHFK_3hs in culture_1
amplified RNA


title: HPV18 immortalized keratinocytes_60hs_1(rep1)


title: HPV18 immortalized keratinocytes_3hs_2(rep2)

description:
HPV18 immortalizes keratinocytes_3hs in culture_2
reference: NHFK_3hs in culture_1
amplified RNA

tag: HPV18 immortalizes keratinocytes


title: HPV18 immortalized keratinocytes_60hs_2(rep1)


title: HPV18 immortalized keratinocytes_3hs_1_TNF(rep2)

description:
HPV18 immortalizes keratinocytes_3hs TNF_1
reference: NHFK_3hs in culture_1
amplified RNA

tag: HPV18 immortalized keratinocytes TNF 3hs


title: HPV18 immortalized keratinocytes_3hs_2(rep1)


title: normal human keratinocytes_3hs_2_TNF(rep2)

description:
normal human keratinocytes_3hs TNF_2
reference: NHFK_3hs in culture_1
amplified RNA

tag: normal human keratinocytes TNF 3hs


title: HPV18 immortalized keratinocytes_60hs_2_TNF(rep2)


title: HPV18 immortalized keratinocytes_3hs_1(rep2)

description:
HPV18 immortalizes keratinocytes_3hs in culture_1
reference: NHFK_3hs in culture_1
amplified RNA


title: HPV18 immortalized keratinocytes_3hs_2_TNF(rep1)


title: HPV18 immortalized keratinocytes_3hs_1(rep1)


title: HPV18 immortalized keratinocytes_3hs_1_TNF(rep1)


title: STROMA_NOR_1

status: Public on Dec 19 2006

type: RNA

city: Ann Arbor

first: Scott

last: Tomlins

state: MI

address: 1400 E. Medical Center Dr., 5410 CCGC

department: Pathology

country: USA

institute: University of Michigan

description: See MIAME checklist, included in the Supplementary Methods, for additional information

hyb_protocol: For hybridization, 40 ug of human Cot-1 DNA (Invitrogen, Carlsbad, CA) was added to 120 ?l of labeled cDNA, the probe was transferred to a Microcon YM-30 filter and centrifuged at 13,000 rcf for 6 min at RT. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The eluted probe volume was adjusted to 18.6 ?l with nuclease free water and the following were added: 4 ?l of yeast tRNA (10 ?g/?l, Invitrogen, Carlsbad, CA), 4.9 ?l of 20X SSC and 0.84 ?l of 10% SDS. The probe was denatured at 100 Co for 3 min and centrifuged at 13,000 rcf for 45 sec. The probe was added directly to the microarray and a cover slip was added. Slides were hybridized overnight in DIE-TECH 1 or 5 slide hybridization chambers in a 65 Co water bath. After hybridization, cover slips were removed by incubation in 2x SSC / 0.05% SDS. Slides were then washed in 2x SSC / 0.05% SDS for 5 min and 0.2x SSC / 0.05% SDS for five minutes. Slides were then washed in 0.2x SSC for 10 sec and centrifuged dry at 500 rpm for 5 min.

scan_protocol: Microarrays were scanned using a GenePix 4000B scanner (Axon Instruments, Union City, CA).

data_processing: Images were gridded and spots were quantified using GenePix Pro 4.0 software (Axon Instruments, Union City, CA).  For all hybridizations, the log2 normalized Median of Ratios (as described below) was used.

molecule: total RNA

label_protocol: Total RNA (~10ng) in a volume of 25 µl was converted into an OmniPlex WTA cDNA library and amplified by WTA PCR using reagents and protocols according to the beta-commercial TransPlex WTA kit (Rubicon Genomics, Ann Arbor, MI). For each sample, a single 10 µl aliquot of the WTA cDNA library was amplified by WTA PCR and products were purified. Four or five 5 ng aliquots of product were subjected to a second WTA PCR amplification in the presence of amino-allyl dUTP for post amplification labeling and products were pooled before proceeding with the hybridization. Yields after all WTA PCR amplifications were between 2 to 5 ?g per reaction.  For all WTA amplified cDNA samples with amino-allyl dUTP incorporated (10-20 ?g), nuclease free water was added to 500 ?l, the sample was transferred to a Microcon YM-30 filter, and centrifuged at 13,000 rcf at RT for 12 minutes. The spin column was inverted in a new collection tube and centrifuged at 13,000 rcf for 2 min at RT. The amount of sample was measured and the volume was adjusted to 5 ?l with nuclease free water. For labeling, 5 ?l of 1 M sodium bicarbonate (pH 9.0) was added and allowed to incubate at RT for 15 min. Nine ?l of anhydrous DMSO were added to Mono Reactive Cy3 and Cy5 dye packs (Amersham, Buckinghamshire, England), mixed thoroughly, and 1 ?l of the proper dye was added to each sample. The labeling mixture was incubated in the dark at RT for 1 hour. The reaction was stopped by the addition of 9 ?l of 4 M Hydroxylamine for 15 min. For each labeling mixture, RNase free water was added to 100 ?l, 500 ?l of PB buffer was added and Cy3 and Cy5 mixtures were added to separate Qiaquick (Qiagen, Valencia, CA) spin columns and centrifuged at 13,000 rcf. Columns were washed twice with 700 ?l of buffer PE, and centrifuged dry at 13,000 rcf for 2 min. To elute, 60 ?l of EB buffer was placed on the column, incubated for 5 min and centrifuged at 13,000 rcf for 1 min. The Cy3 labeled and Cy5 labeled cDNA for each hybridization were mixed and 1.5 ?l were used for analysis on a ND-1000 spectrophotometer.

extract_protocol: Laser Capture Microdissection (LCM) was performed from frozen tissue sections with the SL Microtest device using µCUT software MMI). Approximately 10,000 cells were captured for each sample. Serial sections were used if cells could not be obtained from a single section. Total RNA was isolated from captured cells with the RNAqueous Micro kit (Ambion) and treated with DNAse I according to the manufacturer's instructions. RNA quantification was perfomed using Ribogreen (Molecular Probes).

tag: Normal Stroma - Organ Donor Sample 1

source_name: CPP

extract_protocol: commercially-obtained

tag: Clontech Prostate Pool


title: STROMA_EPIBPH_1

tag: BPH Stroma - Epithelial BPH Sample 1


title: STROMA_BPH_4

tag: BPH Stroma - Stromal BPH nodule Sample 4


title: STROMA_ADJ_PCA_2

tag: Normal Stroma - Adjacent Sample 2


title: STROMA_NOR_2

tag: Normal Stroma - Organ Donor Sample 2


title: STROMA_EPIBPH_2

tag: BPH Stroma - Epithelial BPH Sample 2


title: STROMA_NOR_3

tag: Normal Stroma - Organ Donor Sample 3


title: STROMA_EPIBPH_6

tag: BPH Stroma - Epithelial BPH Sample 6


title: STROMA_BPH_5

tag: BPH Stroma - Stromal BPH nodule Sample 5


title: PIN_13

tag: Prostatic Intraepithelial Neoplasia Sample 13


title: PIN_5

tag: Prostatic Intraepithelial Neoplasia Sample 5


title: PIN_8

tag: Prostatic Intraepithelial Neoplasia Sample 8


title: STROMA_BPH_7

tag: BPH Stroma - Stromal BPH nodule Sample 7


title: PIN_10

description: See MIAME Checklist

tag: Prostatic Intraepithelial Neoplasia Sample 10


title: PIN_7

tag: Prostatic Intraepithelial Neoplasia Sample 7


title: PIN_12

tag: Prostatic Intraepithelial Neoplasia Sample 12


title: PIN_6

tag: Prostatic Intraepithelial Neoplasia Sample 6


title: PIN_9

tag: Prostatic Intraepithelial Neoplasia Sample 9

tag: Clontech Prostate Pool


title: PIN_3

tag: Prostatic Intraepithelial Neoplasia Sample 3


title: STROMA_ADJ_PCA_1

tag: Normal Stroma - Adjacent Sample 1


title: STROMA_EPIBPH_3

tag: BPH Stroma - Epithelial BPH Sample 3


title: PIN_4

tag: Prostatic Intraepithelial Neoplasia Sample 4


title: PIN_11

tag: Prostatic Intraepithelial Neoplasia Sample 11


title: PIN_2

tag: Prostatic Intraepithelial Neoplasia Sample 2


title: PIN_1

tag: Prostatic Intraepithelial Neoplasia Sample 1


title: PCA_26

tag: Prostate Carcinoma Sample 26


title: PCA_30a

tag: Prostate Carcinoma Sample 30a


title: PCA_25

tag: Prostate Carcinoma Sample 25


title: PCA_30b

tag: Prostate Carcinoma Sample 30b


title: PCA_27

tag: Prostate Carcinoma Sample 27


title: PCA_13

tag: Prostate Carcinoma Sample 13


title: PCA_29

tag: Prostate Carcinoma Sample 29


title: PCA_11b

tag: Prostate Carcinoma Sample 11b


title: PCA_22

tag: Prostate Carcinoma Sample 22


title: PCA_17

tag: Prostate Carcinoma Sample 17

tag: Clontech Prostate Pool


title: PCA_10

tag: Prostate Carcinoma Sample 10


title: PCA_21

tag: Prostate Carcinoma Sample 21


title: PCA_12

tag: Prostate Carcinoma Sample 12


title: PCA_14

tag: Prostate Carcinoma Sample 14


title: PCA_28

tag: Prostate Carcinoma Sample 28

tag: Clontech


title: PCA_20

tag: Prostate Carcinoma Sample 20


title: PCA_23

tag: Prostate Carcinoma Sample 23


title: PCA_24

tag: Prostate Carcinoma Sample 24


title: PCA_11a

tag: Prostate Carcinoma Sample 11a


title: PCA_19

tag: Prostate Carcinoma Sample 19

tag: Clontech Prostate pool


title: PCA_7

tag: Prostate Carcinoma Sample 7


title: PCA_18

tag: Prostate Carcinoma Sample 18


title: PCA_15

tag: Prostate Carcinoma Sample 15


title: PCA_9

tag: Prostate Carcinoma Sample 9


title: PCA_8

tag: Prostate Carcinoma Sample 8

tag: Clontech Prostate Pool


title: MET_HR_3b

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 3b


title: MET_HR_7

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 7


title: MET_HR_3a

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 3a


title: PCA_4

tag: Prostate Carcinoma Sample 4


title: MET_HR_4

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 4


title: MET_HR_10

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 10


title: MET_HR_6

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 6


title: MET_HR_13

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 13


title: PCA_3

tag: Prostate Carcinoma Sample 3


title: MET_HR_8

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 8


title: MET_HR_15

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 15


title: MET_HR_2

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 2


title: PCA_6

tag: Prostate Carcinoma Sample 6


title: PCA_16

tag: Prostate Carcinoma Sample 16

tag: Clontech Prostate pool


title: MET_HR_12

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 12


title: MET_HR_11

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 11

tag: Clontech Prostate Pool


title: MET_HR_14

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 14


title: PCA_2

tag: Prostate Carcinoma Sample 2


title: MET_HN_3

tag: Metastatic Prostate Carcinoma - Hormone Naïve Sample 3


title: PCA_1

tag: Prostate Carcinoma Sample 1


title: MET_HR_5

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 5


title: MET_HR_1

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 1


title: MET_HN_2

tag: Metastatic Prostate Carcinoma - Hormone Naïve Sample 2


title: MET_HR_9

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 9


title: PCA_5

tag: Prostate Carcinoma Sample 5


title: MET_HN_1

tag: Metastatic Prostate Carcinoma - Hormone Naïve Sample 1


title: EPI_NOR_1

tag: Normal Prostate Epithelium - Organ Donor Sample 1


title: EPI_NOR_3

tag: Normal Prostate Epithelium - Organ Donor Sample 3


title: EPI_BPH_2

tag: BPH Prostate Epithelium - Epithelial BPH Sample 2


title: EPI_ADJ_PCA_14

tag: Normal Prostate Epithelium - Adjacent Sample 14


title: EPI_BPH_4

tag: BPH Prostate Epithelium - Epithelial BPH Sample 4


title: EPI_NOR_2

tag: Normal Prostate Epithelium - Organ Donor Sample 2


title: EPI_BPH_1

tag: BPH Prostate Epithelium - Epithelial BPH Sample 1

tag: Clontech Prostate Pool


title: EPI_ATR_1

tag: Atrophic Prostate Epithelium - Simple Cystic Atrophy Sample 1


title: EPI_ATR_PIA5

tag: Atrophic Prostate Epithelium (PIA) Sample 5


title: EPI_ATR_2

tag: Atrophic Prostate Epithelium - Simple Atrophy Sample 2


title: EPI_ATR_PIA4

tag: Atrophic Prostate Epithelium (PIA) Sample 4


title: EPI_ADJ_PCA_9

tag: Normal Prostate Epithelium - Adjacent Sample 9


title: EPI_ADJ_PCA_8

tag: Normal Prostate Epithelium - Adjacent Sample 8


title: EPI_ADJ_PCA_3

tag: Normal Prostate Epithelium - Adjacent Sample 3


title: EPI_ADJ_PCA_15

tag: Normal Prostate Epithelium - Adjacent Sample 15


title: EPI_ADJ_PCA_7

tag: Normal Prostate Epithelium - Adjacent Sample 7


title: EPI_ADJ_PCA_13

tag: Normal Prostate Epithelium - Adjacent Sample 13


title: EPI_ADJ_PCA_12

tag: Normal Prostate Epithelium - Adjacent Sample 12


title: EPI_ADJ_PCA_6

tag: Normal Prostate Epithelium - Adjacent Sample 6


title: EPI_ATR_PIA3

tag: Atrophic Prostate Epithelium (PIA) Sample 3


title: Growth stage control_7hr

status: Public on Jul 24 2008

city: St. Paul

first: Zhigang

last: Zhang

state: MN

address: 1479 Gortner Avenue, Suite 140

department: BioTechnology Institute

institute: University of Minnesota

description: Transcriptional profiling of Synechocystis sp. PCC 6803 was compared between time-point samples (7hr) during the normal growth in BG-11 medium  and mid-exponential growth phase cell samples (OD730nm=0.6)

hyb_protocol: Microarray hybridization and washing conditions were based on E. coli cDNA microarray protocols (Khodursky, Bernstein et al. 2003) with some modifications. To reduce cross-hybridization, a pre-hybridization step was performed for Synechocystis slides as follows: 45ml pre-hybridization solution (containing 4.5 ml of 20× SSC, 1.1 ml of HEPES (pH 7), 250 μl of salmon sperm DNA and 250 μl of BSA) was preheated to 65 oC in a water bath. Then microarray slides were incubated vertically in the preheated solution for 45 min at 65 oC. The slides were washed 3 times in sterile dH2O and were spin-dried for 2 min at 600 rpm. The slides were then immediately used for hybridization. The hybridization mixture (total volume 21 μl) contained on-column cleaned Cy3-dUTP and Cy5-dUTP labelled samples, 3 μl of 20×SSC, 0.5 μl of 1M HEPES buffer (pH 7), 2 μl of BSA and 2 μl of salmon sperm DNA as blocking agent and 0.75 μl of 5% SDS. The hybridizations were carried out under a 20×20 flat cover-slip in hybridization chambers (Monterey Industries) submerged in a 65°C water bath for 5 to 6 hours. Slides were washed sequentially in each of the following solutions: Chamber 1: 330 ml of dH2O, 15 ml of 20× SSC, 10 ml of 10% SDS; Chamber 2: 350 ml of dH2O and 1 ml of 20× SSC; and Chamber 3: 350 ml of dH2O and 0.5 ml of 20× SSC. After the washes, the slides were air dried by centrifugation for 3 min at 600 rpm.

scan_protocol: Scanned on an Axon GenePix 4000B laser scanner (Molecular Devices, Sunnyvale, CA) at the resolution of 10 µm per pixel. The scanned 16-bit TIFF images were analyzed by using the software GenePix Pro 4.0 (Molecular Devices, Sunnyvale, CA).

data_processing: The original fluorescence intensity gene expression data were pre-processed using lowess smoothing and an ANOVA linear model. The data were centered. The normalized Cy-5/Cy-3 log2(ratio) values were taken to reflect the relative gene expression level changes. The statistically significant genes were identified by using significance analysis of microarray (SAM) (Tusher et al. 2001) with a false discovery rate lower than 1%.

organism: Synechocystis sp. PCC 6803

source_name: Control sample (mid-exponential phase cells)

label_protocol: Direct fluorescent labelling of reverse-transcribed cDNA was performed. The labelling reaction consisted of 8-15 μg of purified total RNA, 5-10 μg of random hexamer (pdN6), 3μl of dNTP mix (1 mM dATP, 1 mM dCTP, 1 mM dGTP and 0.4 mM dTTP), 1.0 µl of 1mM Cy3- or Cy5-dUTP, 1× superscript buffer, 3.0 µl of 0.1M DTT, and 1µl Superscript II (Invitrogen). The reactions were incubated at room temperature for 10min before transferring to 42oC for 1hr 50min. labeled cDNA was purified using a Microcon YM-30 column (Millipore).

extract_protocol: The bacterial samples were fixed by a stop solution (see above) and centrifuged at 4oC. The cell pellets were snap-frozen in liquid nitrogen before storing at -80oC until required. Total RNA was isolated using standard hot phenol-chloroform method with 1×TE sucrose buffer (10mM Tris, 1mM EDTA, 0.5M sucrose, pH 8) at 50oC instead of using TE buffer (10mM Tris, 1mM EDTA, pH 8) at 25oC. Residual DNA was removed by DNase I digestion.

tag: Control cells (mid-log, 0hr)

source_name: Growth stage control sample

tag: Growth stage control_7hr


title: Exp 3 TP7 (36 hr)

status: Public on Oct 31 2006

city: College Station

first: Gus

middle: John

last: Menger

state: TX

department: Anatomy and Neurobiology

institute: Texas A&M Health Science Center

description: To screen for output signals that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared relative to SCN2.2 cells in vitro and the rat SCN in vivo. Similar to the circadian profiling of the SCN2.2 and rat SCN transcriptomes, NIH/3T3 fibroblasts exhibited rhythmic fluctuations in the expression of the core clock genes, Per2, Bmal1, and Cry1 and 323 functionally-diverse transcripts (2.6%), many of which regulate cellular communication. Overlap in rhythmically-expressed transcripts among NIH/3T3 fibroblasts, SCN2.2 cells and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared to NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among genes mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase (Nos) in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in co-cultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that the gaseous neurotransmitter nitric oxide may play a key role in SCN pacemaker function. Thus, comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective analysis of circadian signals involved in the coupling of SCN oscillators and regulation of rhythmicity in downstream cells.

hyb_protocol: Fragmented biotinylated cRNA (15 µg) was hybridized on Affymetrix GeneChip mouse U74 version 2 arrays at 45°C and 60 rpm in a GeneChip Hybridization Oven 640 (Affymetrix, Santa Clara, CA) for 16 h.

scan_protocol: Following hybridization, arrays were washed and stained using Affymetrix protocols for antibody amplification staining on a GeneChip Fluidics Station 400 in conjunction with Affymetrix Microarray Suite 5.0 software. After a brief wash with a nonstringent buffer, the stained signals on the array were then amplified with a solution containing 3 µg/ml antistreptavidin biotinylated antibody (Vector Laboratories, Burlingame, CA), 1x morpholine ethane sulfonic buffer, 2 mg/ml acetylated BSA, and 0.1 mg/ml normal goat IgG for 10 min followed by a second staining with streptavidin phycoerythrin for 10 min at 25°C. After a final wash with a stringent buffer, the probe array was scanned at the excitation wavelength of 570 nm using an Agilent GeneArray Scanner (Palo Alto, CA). After scanning, each image was first checked for major chip defects or abnormalities during hybridization as a quality control. Arrays were scanned using a global scaling strategy in which the average absolute signal intensity of all arrays was set to an arbitrary target signal intensity of 500 prior to uploading into GeneSpring 7.3 software (Agilent Technologies, Palo Alto, CA).

data_processing: GeneChip signal intensity data from three biological replicates of NIH/3T3 cells were uploaded into GeneSpring 7.2 for additional processing/filtering [Physiol Genomics. 2007 May 11;29(3):280-9]

label: Biotin

organism: Mus musculus

source_name: NIH/3T3 cells

label_protocol: Prior to analysis, the quality of all NIH/3T3 RNA samples was assessed by electrophoresis on 1% agarose gels containing 0.1 µg/ml ethidium bromide. Labeled cRNAs were produced from purified RNA collected at each time point and hybridized on mouse U74v2 GeneChips. Experimental procedures including double-stranded cDNA synthesis and biotinylated cRNA preparation were conducted according to recommended protocols described in Affymetrix GeneChip Expression Analysis Technical Manual. To assure the quality of labeling and fragmentation efficiency, unfragmented and fragmented cRNA products were analyzed on an Agilent 2100 bioanalyzer prior to hybridization on arrays.

growth_protocol: For each of three biological replicates, NIH/3T3 cells (passages 4 and 5) were seeded on culture dishes (60 mm; Corning, Corning, NY) and maintained at 37°C and 5% CO2 in Dulbecco's minimum essential medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum (FBS; HyClone, Logan, UT), 3,000 µg/ml glucose and 292 µg/ml L-glutamine. At 48-h intervals, the medium was changed and cultures were expanded 1:3 or 1:4. Prior to experimental analysis, cultures were plated in multiple T-75 flasks. At ~24 and 44 h after plating, the culture medium was changed so as to respectively lower the FBS concentration to 10% and then to 5%. To facilitate cell cycle and circadian oscillation synchronization across cultures, cells were subjected to medium replacement and exposed to serum-free medium (DMEM) containing 15 µM forskolin (Calbiochem, La Jolla, CA) for 2 h. Cultures were rinsed and thereafter maintained in serum-free DMEM.

extract_protocol: Immediately after forskolin treatment, cells were harvested from individual flasks (approximate density: 1.3 x 106 cells/cm2) at 6-h intervals for 48 h and total cellular RNA was extracted using RNeasy Midi-Kit protocols (Qiagen, Valencia, CA). RNA extracts from individual samples were treated with on-column DNase-I digestion and concentrated with ethanol precipitations.

tag: forskolin pulsed


title: Exp 3 TP6 (30 hr)


title: Exp 3 TP1 (0 hr)


title: Exp 3 TP4 (18 hr)


title: EPI_ADJ_PCA_4

tag: Normal Prostate Epithelium - Adjacent Sample 4

tag: Clontech Prostate Pool


title: EPI_ADJ_PCA_5

tag: Normal Prostate Epithelium - Adjacent Sample 5


title: Exp 3 TP2 (6 hr)


title: MET_HR_16

tag: Metastatic Prostate Carcinoma - Hormone Refractory Sample 16


title: Exp 3 TP5 (24 hr)


title: Exp 3 TP8 (42 hr)


title: Exp 3 TP9 (48 hr)


title: Exp 2 TP9 (48 hr)


title: Exp 2 TP7 (36 hr)


title: Exp 2 TP5 (24 hr)


title: Exp 2 TP8 (42 hr)


title: EPI_ADJ_PCA_11

tag: Normal Prostate Prostate Epithelium - Adjacent Sample 11

tag: Clontech prostate pool


title: EPI_ADJ_PCA_2

tag: Normal Prostate Prostate Epithelium - Adjacent Sample 2


title: EPI_ADJ_PCA_10

tag: Normal Prostate Prostate Epithelium - Adjacent Sample 10

tag: Clontech Normal Prostate pool


title: Exp 2 TP4 (18 hr)


title: Exp 2 TP6 (30 hr)


title: Medium donor 1

status: Public on Nov 10 2006

city: Stanford

last: Stanford Microarray Database

state: CA

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

institute: Stanford Microarray Database (SMD)

description:
Simple annotation: Hematopoiesis, Normal tissue
Image: http://smd.stanford.edu/MicroArray/gifs/2004-07/53414.gif
Compound Based Treatment: 0

hyb_protocol: not provided

scan_protocol: not provided Feature Extraction; Software and parameters for feature extraction.; Protocol Type = Feature Extraction; Parameter Image Origin = 0, 0; Parameter Datafile type = GenePix Results 3; Software: type: feature extraction; Performer: Einar Andreas,,Sivertsen Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Agilent Technologies Scanner; Parameter Pixel Size = 10; Parameter Scan Time = 11:35:30; Parameter Scanner Model = G2505B; Parameter Scanning software version = A.6.3.1; Parameter Scanning software = ChipScan; Parameter Scan Date = 2004-04-27; Performer: Einar Andreas,,Sivertsen Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Agilent Technologies Scanner; Parameter Pixel Size = 10; Parameter Scan Time = 11:35:30; Parameter Scanner Model = G2505B; Parameter Scanning software version = A.6.3.1; Parameter Scanning software = ChipScan; Parameter Scan Date = 2004-04-27; Performer: Einar Andreas,,Sivertsen

data_processing: VALUE is Log (base 2) of the ratio of the median of Channel 2 (usually 635 nm) to Channel 1 (usually 532 nm)

source_name: Amplified Universal Human Reference RNA (Stratagene)

label_protocol: not provided

extract_protocol: not provided

source_name: Amplified RNA from early erythroid progenitors cultured in medium


title: Exp 2 TP1 (0 hr)


title: Exp 2 TP3 (12 hr)


title: Exp 1 TP8 (42 hr)


title: EPI_ADJ_PCA_1

tag: Normal Prostate Epithelium - Adjacent Sample 1

tag: Clontech normal prostate pool


title: Exp 1 TP4 (18 hr)

tag: forskolin pulsed NIH/3T3 cells


title: Exp 1 TP6 (30 hr)


title: EPI_BPH_3

tag: BPH Prostate Epithelium - Epithelial BPH Sample 3


title: Exp 1 TP7 (36 hr)


title: Exp 1 TP5 (24 hr)


title: Corneal Myofibroblast-3

status: Public on Oct 27 2004

city: Baltimore

first: Shukti

last: Chakravarti

state: MD

address: 720 Rutland Ave

department: Medicine

institute: Johns Hopkins University School of Medicine

description:
Corneal stromal cells were isolated from 7-8 weeks old CD-1 mouse and cultured in DMEM containing 10% fetal bovine serum and 10ng/ml TGF-bate 1 as described in our paper. Total RNA of the primary cultured myofibroblasts was extracted using TRIzol reagent. Targets were produced using standard Affymetrix procedures from 5ug of Total RNA. The expression values of all probe sets are scaled to 150 of Target Intensity by using Affymetrix Microarray Suite 5.0. Three replicates of corneal myofibroblasts were analyzed.
Keywords = corneal stroma	myofibroblast	TGF beta 1

hyb_protocol: using standard Affymetrix procedures

scan_protocol: using standard Affymetrix procedures

data_processing:
The model-fitting DNA chip-analyzer software (dChip; Affymetrix Inc.) and an invariant-set normalization method were used for data normalization and to calculate a model-based expression value for each transcript.
The data table below reports both the MAS5.0- and dChip-calculated data.

source_name: cultured corneal stromal cells


title: Exp 1 TP1 (0 hr)

label: biotin

source_name: NIH/3T3 fibroblasts, forskolin-pulsed

tag: NIH/3T3 cells


title: Corneal Myofibroblast-2

label_protocol: Targets were produced using standard Affymetrix procedures from 5ug of Total RNA.

extract_protocol: Total RNA of the primary cultured myofibroblasts was extracted using TRIzol reagent.


title: Corneal Fibroblast-3

description:
Corneal stromal cells were isolated from 7-8 weeks old CD-1 mouse and cultured in DMEM containing 10% fetal bovine serum as described in our paper. Total RNA of the primary cultured fibroblasts was extracted using TRIzol reagent. Targets were produced using standard Affymetrix procedures from 5ug of Total RNA. The expression values of all probe sets are scaled to 150 of Target Intensity by using Affymetrix Microarray Suite 5.0. Three replicates of corneal fibroblast were analyzed.
Keywords = corneal stroma	fibroblast


title: Corneal Fibroblast-1

label_protocol: Targets were produced using standard Affymetrix procedures from 5ug of total RNA.

extract_protocol: Total RNA of the primary cultured fibroblasts was extracted using TRIzol reagent.


title: Corneal Keratocyte-3

description:
Corneal stromal cells were isolated from 7-8 weeks old CD-1 mouse and cultured in DMEM containing 1% platelet-poor horse serum as described in our paper. Total RNA of the primary cultured keratocytes was extracted using TRIzol reagent. Targets were produced using standard Affymetrix procedures from 5ug of total RNA. The expression values of all probe sets are scaled to 150 of Target Intensity by using Affymetrix Microarray Suite 5.0. Three replicates of corneal keratocytes were analyzed.
Keywords = corneal stroma	fibroblast


title: Corneal Fibroblast-2


title: mouse P10 Cornea-1

description:
Corneal stroma was dissected from postnatal 10 days CD-1 mouse. Total RNA was extracted using TRIzol reagent.Targets were produced using standard Affymetrix procedures from 5ug of total RNA. The expression values of all probe sets are scaled to 150 of Target Intensity by using Affymetrix Microarray Suite 5.0. Two replicates of P10 corneal stroma were analyzed.
Keywords = cornea	maturation

source_name: corneal stroma


title: Exp 2 TP2 (6 hr)


title: Corneal Keratocyte-1

extract_protocol: Total RNA of the primary cultured keratocytes was extracted using TRIzol reagent.


title: mouse Adult Cornea-2

description:
Corneal stroma was dissected from 7-8 weeks old CD-1 mouse. Total RNA was extracted using TRIzol reagent. Targets were produced using standard Affymetrix procedures from 5ug of total RNA. The expression values of all probe sets are scaled to 150 of Target Intensity by using Affymetrix Microarray Suite 5.0. Three replicates of adult corneal stroma were analyzed.
Keywords = cornea	maturation


title: mouse Adult cornea-1

hyb_protocol: using standard Affymetrix protocol

scan_protocol: using standard Affymetrix protocol

extract_protocol: Total RNA was extracted using TRIzol reagent.


title: mouse P10 Cornea-2


title: mouse Adult Cornea-3


title: Corneal Keratocyte-2


title: Exp 1 TP9 (48 hr)


title: Exp 1 TP2 (6 hr)


title: Corneal Myofibroblast-1

description:
Corneal stromal cells were isolated from 7-8 weeks old CD-1 mouse and cultured in DMEM containing 10% fetal bovine serum and 10ng/ml TGF-bate 1 as described in our paper. Total RNA of the primary cultured myofibroblasts was extracted using TRIzol reagent.  Targets were produced using standard Affymetrix procedures from 5ug of Total RNA. The expression values of all probe sets are scaled to 150 of Target Intensity by using Affymetrix Microarray Suite 5.0. Three replicates of corneal myofibroblasts were analyzed.
Keywords = corneal stroma	myofibroblast	TGF beta 1


title: LW01set2_mpk3_Psm_24h

status: Public on Jul 18 2008

city: St Paul

first: lin

last: wang

address: 350 Cargill building, 1500 Gortner Ave

department: Plant Biology

description: mpk3 infiltrated with 1x10E8 cfu/ml Pseudomonas syringae ES4326, harvested 24h after infiltration in set2 experiment.

hyb_protocol: Labeled RNA was suspended in hybridization buffer (50% Formamide, 5xSSC, 0.1% SDS, 10 ug Sheared Salmon Sperm DNA (Eppendorf), 50pg calbation oligo)

scan_protocol: Slide was scaned using Genepix4000B with low and high voltages of photomultiplier at 532nm and constant voltage at 635nm.

data_processing:
Note that each Sample corresponds to two GPR files (we use low PMT and high PMT, then combine them).

The median of ratios for each spot was processed by the following linear model: Sij = mu + Ai + Bj + Eij where Sij denotes the log2-transformed, median of ratios for the spot, mu denotes a constant, Ai and Bj denote the effects of i-th gene and j-th subarray. The residual Eij is assumed to be independent and normally distributed. Stable genes-based quantile normalization was applied for slide-to-slide comparisons. LW01_set1_Col-0_Psm_24h, LW01_set1_eds5_Psm_24h, LW01_set1_mpk3_Psm_24h, LW01_set1_mpk6_Psm_24h, LW01_set1_npr_Psm_24h, LW01_set1_pad4_Psm_24h, LW01_set1_sid2_Psm_24h, LW01_set1_tga_Psm_24h, LW01_set2_Col-0_Psm_24h, LW01_set2_eds5_Psm_24h, LW01_set2_mpk3_Psm_24h, LW01_set2_mpk6_Psm_24h, LW01_set2_npr_Psm_24h, LW01_set2_pad4_Psm_24h, LW01_set2_sid2_Psm_24h, LW01_set2_tga_Psm_24h, LW01_set3_Col-0_Psm_24h, LW01_set3_eds5_Psm_24h, LW01_set3_mpk3_Psm_24h, LW01_set3_mpk6_Psm_24h, LW01_set3_npr_Psm_24h, LW01_set3_pad4_Psm_24h, LW01_set3_sid2_Psm_24h, LW01_set3_tga_Psm_24h, LW02_set1_Col-0_Psm_24h, LW02_set1_SALK023199_Psm_24h, LW02_set1_SALK066990_Psm_24h, LW02_set1_coi1_Psm_24h, LW02_set1_dde2_Psm_24h, LW02_set1_ein2_Psm_24h, LW02_set1_jai1_Psm_24h, LW02_set1_jar1_Psm_24h, LW02_set2_Col-0_Psm_24h, LW02_set2_SALK023199_Psm_24h, LW02_set2_SALK066990_Psm_24h, LW02_set2_coi1_Psm_24h, LW02_set2_dde2_Psm_24h, LW02_set2_ein2_Psm_24h, LW02_set2_jai1_Psm_24h, LW02_set2_jar1_Psm_24h, LW02_set3_Col-0_Psm_24h, LW02_set3_SALK023199_Psm_24h, LW02_set3_SALK066990_Psm_24h, LW02_set3_coi1_Psm_24h, LW02_set3_dde2_Psm_24h, LW02_set3_ein2_Psm_24h, LW02_set3_jai1_Psm_24h, LW02_set3_jar1_Psm_24h, LW03_set1_Col-0_Psm_24h, LW03_set1_Col-0_mock_24h, LW03_set1_SALK018487_Psm_24h, LW03_set1_SALK052422_Psm_24h, LW03_set1_SALK084141_Psm_24h, LW03_set1_SALK143063_Psm_24h, LW03_set1_eds1_Psm_24h, LW03_set1_fmo1_Psm_24h, LW03_set2_Col-0_Psm_24h, LW03_set2_Col-0_mock_24h, LW03_set2_SALK018487_Psm_24h, LW03_set2_SALK052422_Psm_24h, LW03_set2_SALK084141_Psm_24h, LW03_set2_SALK143063_Psm_24h, LW03_set2_eds1_Psm_24h, LW03_set2_fmo1_Psm_24h, LW03_set3_Col-0_Psm_24h, LW03_set3_Col-0_mock_24h, LW03_set3_SALK018487_Psm_24h, LW03_set3_SALK052422_Psm_24h, LW03_set3_SALK084141_Psm_24h, LW03_set3_SALK143063_Psm_24h, LW03_set3_eds1_Psm_24h, LW03_set3_fmo1_Psm_24h, LW05_set1_Col-0_Psm-cor-_24h, LW05_set1_Col-0_Psm_24h, LW05_set1_Col-0_mock_24h, LW05_set1_coi1_Psm-cor-_24h, LW05_set1_coi1_Psm_24h, LW05_set1_coi1_mock_24h, LW05_set1_ndr1_Psm_24h, LW05_set1_pbs3_Psm_24h, LW05_set2_Col-0_Psm-cor-_24h, LW05_set2_Col-0_Psm_24h, LW05_set2_Col-0_mock_24h, LW05_set2_coi1_Psm-cor-_24h, LW05_set2_coi1_Psm_24h, LW05_set2_coi1_mock_24h, LW05_set2_ndr1_Psm_24h, LW05_set2_pbs3_Psm_24h, LW05_set3_Col-0_Psm-cor-_24h, LW05_set3_Col-0_Psm_24h, LW05_set3_Col-0_mock_24h, LW05_set3_coi1_Psm-cor-_24h, LW05_set3_coi1_Psm_24h, LW05_set3_coi1_mock_24h, LW05_set3_ndr1_Psm_24h, and LW05_set3_pbs3_Psm_24h were normalized together. Perl scripts for the linear model and the normalization are available for non-commercial research conducted upon request (Fumiaki Katagiri, katagiri@umn.edu). Spots with bad quality (missing, lints, and high background) were flagged as indicated in Flag in the attached .gpr file for this sample.

organism: Arabidopsis thaliana

source_name: Arabidopsis thaliana mpk3, leaves, 1x10E8 cfu/ml Pseudomonas syringae ES4326-infiltrated, 24h

label_protocol: Following procedure was performed with Amino Allyl MessageAmp aRNA Amplification Kit II (Ambion).First, cDNA was synthesized from 1ug of total RNA.  Next, RNA was in vitro transcribed.  The RNA was then labeled with Alexa555 in coupling reaction.

growth_protocol: Plants were grown in pots with BM-2 soil (Berger Peat Moss Ltd, Quebec, Canada) at a density of 9 plants per pot and kept at 22 degrees Celsius and 75% humidity with a 12 hour day length.

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: Individual leaves were infiltrated in the morning using a needle-less syringe with 1x10E8 cfu/ml Pseudomonas syringae ES4326 and harvested 24h later.

tag: Genotype

tag: Tissue

value: leaves

tag: Age

value: 4 week-old

tag: Treatment

tag: Time point


title: NHDF HU

status: Public on Jul 17 2008

city: Cambridge

first: Kate

last: Rubins

state: MA

address: Nine Cambridge Center

institute: Whitehead Institute for Biomedical Research

description:
Simple annotation: Normal tissue, Fibroblasts
Image: http://smd.stanford.edu/MicroArray/gifs/2005-04/60871.gif

scan_protocol: Rubins 2003 - Data filtering and analysis; Array images were scanned using an Axon Scanner 4000A (Axon Instruments, Union City, CA), and image analysis was performed using GenePix Pro version 3.0.6.89 (Axon Instruments, Union City, CA).  Grids were manually aligned and features were found using the software feature extraction.  Features were visually inspected for blemishes and removed from downstream analysis.    Data were uploaded to the Stanford Microarray Database (http://genome-www5.stanford.edu/)  and normalized using regression correlation.  Data were expressed as the log2 ratio of fluorescence intensities of the sample and the reference, for each element on the array.    Data were filtered to exclude elements that did not have a regression correlation of Cy5 to Cy3 signal over the pixels spanning the array element of ??0.6 and intensity/background ratio of ??2.5 in at least 80% of the arrays.    For each gene, the expression levels over the timecourse for each monkey were ??time-zerotransformed?? by subtracting the average of the pre-infection baseline expression level from that animal so that the values of each timepoint represent changes relative to the uninfected samples.    The genes whose expression varied from the uninfected baseline by at least 3 fold in at least 3 samples were selected for further analysis.    The data were hierarchically clustered using the Cluster program (http://rana.lbl.gov/EisenSoftware.htm) and displayed using TreeView (http://rana.lbl.gov/EisenSoftware.htm).; Protocol Type = Feature Extraction; Software: type: feature extraction; Performer: Kate,,Rubins Scanning parameters - Channel 1; Parameters for laser scanning of a hybridized array in channel 1.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 40.01; Parameter Scan Time = 19:26:47; Parameter PMT Volts = 550; Parameter Scan Date = 2005-03-07; Parameter Pixel Size = 10; Parameter Laser on time = 208525; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000A [47263]; Parameter Laser Power = 1.44; Parameter Scan Power = 100; Parameter Scanning software version = 2.0.2.3; Parameter Scanning software = AxImageIO; Performer: Kate,,Rubins Scanning parameters - Channel 2; Parameters for laser scanning of a hybridized array in channel 2.; Protocol Type = Scanning parameters; Parameter Scanner Make = Axon Instruments (V1.00); Parameter Scan Temperature = 40.01; Parameter Scan Time = 19:26:47; Parameter PMT Volts = 620; Parameter Scan Date = 2005-03-07; Parameter Pixel Size = 10; Parameter Laser on time = 573329; Parameter Focus Position = 0; Parameter Scanner Model = GenePix 4000A [47263]; Parameter Laser Power = 2.00; Parameter Scanning software version = 2.0.2.3; Parameter Scan Power = 100; Parameter Scanning software = AxImageIO; Performer: Kate,,Rubins

source_name: Stratagene Universal Human Reference RNA, 1 round of amp(Ambion MessageAmp), 6/27/02

source_name: NHDF HU

label_protocol:
Rubins 2003 - Ambion Message Amp I; FIRST STRAND CDNA SYNTHESIS: In PCR reaction tube, mix: Amount Reagent 0.1 - 5ug  Total RNA 1uL T7 Oligo (dT) primer 11uL Nuclease free water  70oC for 10 min in a thermocycler with lid on, but not clamped down.   Centrifuge briefly.   Prepare in a Master Mix and keep at room temperature: Amount Reagent 2uL 10X First Strand Buffer 1uL Ribonuclease Inhibitor 4uL dNTP Mix Gently pipette or flick to mix, and then centrifuge briefly.  Incubate master mix at 42oC for 2 minutes prior to adding to reaction tubes.   Add to the master mix: Amount Reagent 1uL Reverse Transcriptase  Quickly transfer 8ul of above Master Mix to each RNA sample, mix by pipetting.   42oC for 2 hours in thermocycler with lid on, but not clamped down.   Centrifuge briefly, place on ice, and immediately proceed to next step  SECOND STRAND SYNTHESIS: Amount Reagent 63uL Nuclease free water 10uL 10X Second Strand Buffer 4uL dNTP Mix 2uL DNA Polymerase 1uL RNase H Add 80uL to each tube. Gently pipette or flick to mix, and then centrifuge briefly.  16oC for 2 hours in thermocycler with lid completely off.    DS cDNA CLEANUP: Preheat the 5mL bottle of water at 50oC for at least 10 minutes. Place filter cartridge in a 2mL tube and add 100ul of cDNA Binding Buffer. Incubate at room temperature for 5 min.  Transfer 100uL reaction from strip-8 PCR tubes into 1.5ml Eppendorf tubes. Add 250uL of cDNA binding buffer to each cDNA sample, mix by pipetting. Add the mix to the filter cartridge. Centrifuge for 1 min at 10,000 x g. Discard flow-through.  Add 650ul of cDNA wash buffer. Centrifuge for 1 min. at 10,000 x g. Discard flow-through. Centrifuge again for 1 min at 10,000 x g to remove trace amounts of ethanol.  Transfer filter cartridge to fresh 2mL tube Add 50uL of preheated water to the center of the column.   Incubate at room temperature for 2 minutes. Centrifuge for 1 minute at 10,000 x g. Repeat elution with a second 50ul of preheated water. Centrifuge for 1 minute at 10,000 x g.   Transfer to strip-8 PCR tubes and mark a line for 8uL Concentrate the purified cDNA to < 8uL by drying in a vacuum centrifuge on high heat.   Resuspend cDNA up to 8uL with H20, mix well, and keep on ice or store at ?20oC.  IN VITRO TRANSCRIPTION  Prepare the following master mix: Amount Reagent 8uL T7 NTP mix (75 mM each) 2uL T7 10X Reaction Buffer 2uL T7 Enzyme Mix Add 12uL to each tube. Gently pipette or flick to mix, and then centrifuge briefly.  37oC for 6 hours in hybridization oven.  Add to each reaction: Amount Reagent 2uL DNaseI  Gently pipette or flick to mix, and then centrifuge briefly.  37oC for 30min in hybridization oven   aRNA PURIFICATION  Preheat 5mL bottle of nuclease free water at 50oC for at least 10 minutes.  Immediately before staring aRNA purification., equilibrate 1 filter cartridge per aRNA sample Place filter cartridge in a 2mL tube and add 100ul of aRNA Binding Buffer. Incubate at room temperature for 5 min.  Add 78uL aRNA Elution Solution to each IVT mix to bring final volume up to 100uL.   Add 350uL of aRNA Binding Buffer to each aRNA sample. Add 250uL of ACS grade 95% ethanol to each sample. Mix by pipetting or gentle vortexing.  Apply entire sample to the equilibrated filter cartridge.  Centrifuge for 1 minute at 10,000 x g.   Discard flow-through but re-use tube.  Add 650uL aRNA Wash Buffer to each filter.   Centrifuge for 1 minute at 10,000 x g.   Discard flow-through but re-use tube. Centrifuge again for 1 min at 10,000 x g Transfer cartridge to a fresh 2mL tube.  Elute aRNA by adding 50uL preheated (50oC) water to center of column.  Incubate at room temperature for 2 min.  Centrifuge for 1 minute at 10,000 x g.  Repeat elution with a second 50uL of preheated water. Centrifuge for 1 minute at 10,000 x g.   Store aRNA at ?80oC.; Protocol Type = Amplification; Performer: Kate,,Rubins
Rubins 2003 - Labeling Amplified RNA - Direct Incorporation of Cy Dyes; Preparation of Fluorescent DNA Probe from Amplified mRNA  1. To anneal primer, mix 3-4ug of amplified mRNA with 10ug of random hexamer primer in a total volume of 15.4ul:    mRNA 3-4ug  pdN6 (5 ug/uL) 2uL ddH2O (DEPC) to 15.4 uL Total volume: 15.4 uL  2. Heat to 65oC for 10 min and cool on ice.   3. Add 14.6uL reaction mixture (Master Mix or separate) to each RNA/hexamer mix Reaction mixture  5X first-strand buffer 6.0uL  0.1M DTT 3.0uL Unlabeled dNTPs (low dTTP) 0.6uL Cy3 or Cy5 (1 mM, Amersham) 3.0uL Superscript II (200 U/uL, Gibco BRL) 2.0uL Total volume: 14.6uL   4. Incubate at 42oC for 1 hr.   5. Add 1 uL SSII (RT booster) to each sample. Incubate for an additional 1 hr.   6. Degrade RNA by addition 15uL of 0.1N NaOH and incubate at 65-70oC for 10 min.   7. Neutralize by addition of 15uL of 0.1N HCl.  8. Add 380uL of TE (10mM Tris, 1mM EDTA) to a Microcon YM-30 column.  Next add the 60uL of Cy5 probe and the 60??l of Cy3 probe to the same Microcon.  9. WASH 1: Spin 7-8 min. at 14,000g (=RCF).  Remove flow through and save to a separate tube.  10. WASH 2: Add 450ul 1X TE and spin 7-8 min. at 14,000g. Remove flow through and save to the separate tube.  11.  WASH 3: Add to centricon: 1X TE 450uL 1ug/uL Cot1 human DNA (Gibco-BRL) 20 uL 10ug/uL  polyA RNA (Sigma, #P9403) 2 uL 10ug/uL  tRNA (Gibco-BRL, #15401-011) 2 uL Spin 7-10 min. at 14,000g. Look for nice concentration of the "colored probe" in the centricon. Try to concentrate to a volume of less than 25uL. This volume is attained when the center of the membrane is dry and the probe forms a ring of liquid at the edges of the membrane.  Make sure not to dry the membrane completely!  12. Invert the centricon into a clean tube and spin briefly at 14,000 RPM to recover the probe.; Protocol Type = Labeling; Performer: Kate,,Rubins

extract_protocol: Rubins 2003 - Trizol Extraction of Total RNA from PBMCs; 1. Make sure the area is clean and free of any RNases.  Keep the samples cold or on ice as much as possible to prevent RNA degradation.   Use only RNase free tubes and RNase free, aerosol-barrier pipette tips throughout the whole experiment.  2. Add 1mL of Trizol to the frozen PBMC pellet.   3. If the Trizol was added to the PBMCs before freezing, thaw the Trizol/PBMCs on the benchtop for 5-10 minutes.  4. Vortex and transfer to 1.5ml microfuge tubes if not already in them.  5. Add 0.2 mls chloroform per 1 ml Trizol.  Vortex or shake vigorously and incubate at room temp. for 2-3 minutes.  6. Centrifuge at 12,000g for 15 minutes at 4C.  7. Transfer the aqueous phase to a fresh tube.  (The volume will be about 60% of the volume of Trizol.)  Note:  If isolating DNA, save the organic phase.  Add 1uL of 1ug/uL Linear Acrylamide if RNA yield is expected to be low.  8. Add 0.5 mLs of isopropanol per 1 ml Trizol used.  Incubate at room temp. for 10 minutes.   9. Centrifuge at top speed (14,000rpm) for 10 minutes at 4C.    10. The RNA pellet will be visible at the bottom of the tube.  Use the vacuum to very carefully aspirate the supernatant. Alternatively, carefully suck off the supernatant with a pipette. Discard supernatant. Check that the pellet is visible at the bottom of the tube.  11. WASH 1: Wash the pellet with 1 ml 70% EtOH.  Centrifuge at top speed 14,000g for 5-7 minutes.  12. Use the vacuum to very carefully aspirate the supernatant. Alternatively, carefully suck off the supernatant with a pipette. Discard supernatant. Check that the pellet is visible at the bottom of the tube.  13. WASH 2:  Wash the pellet with 1 ml 70% EtOH.  Centrifuge at top speed 14,000g for 5-7 minutes.  14. Use the vacuum to very carefully aspirate the supernatant. Alternatively, carefully suck off the supernatant with a pipette. Discard supernatant. Check that the pellet is visible at the bottom of the tube.  15. Air dry pellet shortly, until no EtOH is visible (2-5 minutes).  Do not overdry!  16. Resuspend the pellet in 20uls of DEPC H2O.  Store at ?80C.  17. Check RNA quality and quantity by denaturing gel and OD spec (260nm and 260/280 ratio).; Protocol Type = Extract preparation; Performer: Kate,,Rubins

tag: NHDF HU

tag: Compound Based Treatment

value: Hydroxyurea 10mM

tag: Infection

tag: Cell Line

value: Primary Human Fibroblast

tag: Measurement

value: time(relative) 24 h


title: No respiratory symptoms (Viro3P11-172)

status: Public on Jul 09 2008

city: San Francisco

first: Joe

last: DeRisi

department: Biochemistry and Biophysics

institute: University of California, San Francisco

description: RdABC amplified RNA from Nasal Lavage

hyb_protocol: standard overnight hybridization

scan_protocol: Axon 4000 scanner

data_processing: Median feature intensity minus median background intensity. The data analysis software used is E-Predict, which normalizes the data using a variety of measures: sum, quadratic, unit-vector, or no normalization (please see Urisman A, Fischer KF, Chiu CY, Kistler AL, Beck  S, Wang D, DeRisi JL.E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.Genome Biol. 2005;6(9):R78. Epub 2005 Aug 30).  E-Predict is public access.  For the purposes of this paper, sum-normalized intensities were used in the analysis.

organism: Viruses

source_name: RdABC amplified RNA from Nasal Lavage

label_protocol: Rd C Cy3 incorporation

extract_protocol: RNeasy followed by DNAse treatment

tag: No respiratory symptoms