title: Non-Pregnant_Endometrium_Sow, biological replicate 2

description:
Non-Pregnant Endometrium 2
Experimental replicate 2 of 4 (Non-Pregnant Endometrium).

source_name: Mesometrial endometrium from a non-pregnant sow, mid-estrus, sow #2


title: S.coelicolor germination_time 1.5 h_replicate 2

status: Public on Aug 01 2013

type: RNA

city: Prague

first: Jiri

last: Vohradsky

address: Videnska 1083

country: Czech Republic

institute: Institute of Microbiology Czech Academy of Sciences

hyb_protocol: Bacterial RNA Amplification Protocol for Gene Expression by Oxoford Gene Technology

scan_protocol: Scanned on an Agilent Technologies Scanner G2505C and images were quantified using  Agilent Feature Extraction software.

data_processing: For background subtraction and LOWESS normalization was used Agilent Feature Extraction software. The GeneSpring software was used to filter out signal intensities < 200.

molecule: total RNA

organism: Streptomyces coelicolor A3(2)

source_name: S.coelicolor germination time 1.5 h

growth_protocol: Dormant spores germiantion was activated by mechanical disruption and 10min heat shock (50°C), than cultivated in rich AM liquid medium at 37°C. Samples collected at 30 min intervals.

extract_protocol: The cells were mechanicaly disrupted and total RNA was extracted using phenol-chlorophorm RNA extraction.

tag: strain

source_name: proportional mixture of RNA samples from all time points


title: Vector rep3

city: Montréal

first: Mathieu

last: Vernier

state: Quebec

address: 2900 Edouard Montpetit

department: Biochemistry

country: Canada

institute: Université de Montréal

description: Gene expression data from H1299 infected with an empty vector

hyb_protocol: Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on Human Gene 2.0 ST Chips. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.

scan_protocol: GeneChips were scanned using the GeneChip® Scanner 3000 7G/4 Color Early Access.

data_processing: The data were analyzed with the affymetrix Expression Console software using Affymetrix default analysis settings and RMA as normalization method. Transcripts with a fold change higher or equal to 1.5 and a p-value˂ 0.05 according to the two-sample student's t-test were selected for final determination.

label: biotin

organism: Homo sapiens

source_name: H1299 infected with an empty vector, 5 days post-infection

label_protocol: Biotinylated cRNA were prepared according to the protocol from the Ambion® WT Expression kit for Affymettrix® GeneChip®Whole Transcrpit (WT) Expression Arrays

growth_protocol: Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Wisent, Montréal, QC) supplemented with 10% fetal bovine serum (FBS; Wisent) and 1% penicillin G/streptomycin sulfate (Wisent).

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

treatment_protocol: Cells were infected with an empty retrovirus or a retrovirus expressing CHES1 and selected during three days with 5 µg/ml puromycin before RNA extraction.

tag: cell line

tag: vector

value: empty


title: gtl1-3

city: Columbus

first: Kengo

last: Morohashi

state: OH

address: 1060 Carmack Rd

department: Molecular Genetics

country: USA

institute: The Ohio State University

description: gtl1-1 mutant trichome, biological replicate 3

hyb_protocol: Hybridized onto Arabidopsis ATH1 Genome GeneChips® (Affymetrix Inc., USA) according to manifucture's instruction (Affymetrix).

scan_protocol: GeneChips are scanned in a GeneChip Scanner 3000 (Affymetrix Inc., USA) and CEL files are generated from DAT files using GeneChip® Operating Software (GCOS) software (Affymetrix Inc., USA).

data_processing: Affy-GUI, normalized by GCRMA.

organism: Arabidopsis thaliana

source_name: gtl1-1 mutant trichome

label_protocol: RNA probes were labeled using an Affymetrix 3' IVT Express Kit (Affymetrix, Japan)

growth_protocol: Plants were germinated and grown on plates containing Murashige and Skoog salts, pH 5.7, 1% sucrose (w/v), and 0.5% phytogel (w/v) in continuous light at 22°C.

extract_protocol: Trichomes were removed with forceps from the fifth and sixth rosette leaves of three-week-old wild-type and gtl1-1 mutant plants according to a previously established protocol (Jakoby et al, 2008). Total RNA was extracted from three biological replicates by a Qiagen plant RNeasy kit, and treated with DNaseI (Takara Bio, Japan) following the manufacturer’s instructions.

tag: genotype/variation

value: gtl1-1 mutant

tag: tissue

value: trichome


title: S.coelicolor germination_time 0.5 h_replicate 3

source_name: S.coelicolor germination time 0.5 h

label_protocol: Bacterial RNA Amplification Protocol for Gene Expression by Oxoford Gene Technology


title: S.coelicolor germination_time 1 h_replicate 2

source_name: S.coelicolor germination time 1 h


title: Arresting_Trophoblast_gestation_day_20, biological replicate 3

city: Hamilton

first: Jocelyn

middle: M

last: Wessels

state: Ontario

address: 1280 Main Street West

department: Obstetrics and Gynecology

institute: McMaster University

description:
Pig 11 Arresting Trophoblast
Experimental replicate 3 of 3 (Arresting Trophoblast).

hyb_protocol: Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://microrna.sanger.ac.uk/sequences/) or other RNA (control or customer-defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.

scan_protocol: Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).

data_processing:
Microarray assay was performed by LC Sciences.
Background is determined using a regression-based background mapping method. The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots. Raw data matrix is then subtracted by the background matrix.
Normalization is carried out using a LOWESS (Locally-weighted Regression) method on the background-subtracted data. The normalization is to remove system-related variations, such as sample amount variations, different labeling dyes, and signal gain differences of scanners so that biological variations can be faithfully revealed [B. M. Bolstad, R. A. Irizarry, M. Astrandand T. P. Speed, (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias, Bioinformatics, 19 (2), 185-193]. A transcript to be listed as detectable must meet at least two conditions: signal intensity higher than 3×(background standard deviation) and spot CV < 0.5. CV is calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level.
T-test is performed between control and test sample groups. T-values are calculated for each miRNA, and p-values are computed from the theoretical t-distribution. miRNAs with p-values below a critical p-value (typically 0.01) are selected for cluster analysis. The clustering is done using a hierarchical method and is performed with average linkage and Euclidean distance metric.
All data processes, except clustering plot, are carried out using in-house-developed computer programs. The clustering plot is generated using TIGR MeV (Multiple Experimental Viewer) software from The Institute for Genomic Research.

organism: Sus scrofa

source_name: Trophoblast associated with an arresting gestation day 20 conceptus (~50% smaller, and less vascularization than littermates), from sow #11

label_protocol: Microarray assay was performed using a service provider (LC Sciences). The assay started from 2 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore), and the small RNAs (< 300 nt) isolated were 3'-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent Cy3/Cy5 dye staining.

growth_protocol: None. Gestation Day 20 sows (Yorkshire breed).

extract_protocol: Total RNA was extracted using the miRNeasy mini kit (Qiagen).

treatment_protocol: None.

tag: breed

value: Yorkshire

tag: gender

value: female

value: trophoblast

tag: pregnancy status

value: pregnant

tag: status of conceptus attachement site

value: arresting

tag: sow identifier


title: gtl1-1

description: gtl1-1 mutant trichome, biological replicate 1


title: gtl1-2

description: gtl1-1 mutant trichome, biological replicate 2


title: WT-3

description: Wild type trichome, biological replicate 3

source_name: Wild type trichome

value: wild-type


title: WT-2

description: Wild type trichome, biological replicate 2


title: WT-1

description: Wild type trichome, biological replicate 1


title: Vector rep2


title: S.coelicolor germination_time 1 h_replicate 1


title: Healthy_Trophoblast_gestation_day_20, biological replicate 3

description:
Pig 11 Healthy Trophoblast
Experimental replicate 3 of 3 (Healthy Trophoblast).

source_name: Trophoblast associated with a healthy gestation day 20 conceptus, from sow #11

value: healthy


title: Non-Pregnant_Endometrium_Sow, biological replicate 1

description:
Non-Pregnant Endometrium 1
Experimental replicate 1 of 4 (Non-Pregnant Endometrium).

source_name: Mesometrial endometrium from a non-pregnant sow, mid-estrus, sow #1

value: endometrium

value: non-pregnant


title: S.coelicolor germination_time 0 h_replicate 3

description: Samples collected after 10min heat shock (50°C) activation; marked as time 0 h

source_name: S.coelicolor germination time 0 h


title: Arresting_Endometrium_gestation_day_20, biological replicate 3

description:
Pig 11 Arresting Endometrium
Experimental replicate 3 of 3 (Arresting Endometrium).

source_name: Endometrium associated with an arresting gestation day 20 conceptus (~50% smaller, and less vascularization than littermates), from sow #11


title: S.coelicolor germination_time 0.5 h_replicate 1


title: Healthy_Trophoblast_gestation_day_20, biological replicate 2

description:
Pig 12 Healthy Trophoblast
Experimental replicate 2 of 3 (Healthy Trophoblast).

source_name: Trophoblast associated with a healthy gestation day 20 conceptus, from sow #12


title: S.coelicolor germination_time 0.5 h_replicate 2


title: NLPHL_AD_M3

city: Frankfurt

first: Claudia

last: Döring

address: Theodor Stern Kai 7

department: Medical School, Institite of Pathology

country: Germany

institute: Johann Wolfgang-Goethe University Frankfurt a.M.

hyb_protocol: Human Gene 1.0 ST arrays (Affymetrix) were hybridized with 5.5 mg labelled cDNA.

scan_protocol: Affymetrix Gene ChIP Scanner 3000 7G

data_processing:
Statistical analysis was done with the statistical computing environment R [version 2.15.1 (2012-06-22)]. Additional software packages (affy [version 2.10], geneplotter, multtest, and vsn [version 2.10]) were taken from the Bioconductor project.  Microarray preprocessing: Probe level normalization was conducted using the variance stabilization method (vsn) from Huber et al..
probe group file: Unsupported Human Gene 1.0 ST Array CDF Files
meta-probeset file: HuGene-1_0-st-v1 Probeset Annotations, CSV Format, Release 30 (11 MB 01/22/10)

source_name: NLPHL epithelioid cells

label_protocol: Samples were labelled with Encore biotin module (NUGEN) washed and stained using the HWS Kit (Affymetrix).

extract_protocol: RNA was amplified directly from cells using the One-direct-Kit from NUGEN applied for 250 microdissected cells

treatment_protocol: Cells were microdissected from HE stained kryosections

tag: cell type

value: NLPHL epithelioid cells


title: P2F30_1 with array type poplar from Affymetrix

city: evry

first: stephanie

last: huguet

address: 2 rue gaston cremieux

country: France

institute: inra

description:
populus tremula x populus alba  - harvest date: 18-04-11
genes regulated by PtaZFP2 in absence of mechanical stress - genes regulated by PtaZFP2 after one bending.Species: Populus tremula x Populus alba

hyb_protocol: labelled extract quantity: 15ug

scan_protocol: GCOS, Biotin:pmt voltage 570nm,650V,laser power 1

data_processing: The data were normalized with the gcrma algorithm (Irizarry et al., 2003), available in the Bioconductor package (Gentleman and Carey, 2002).

label: Biotin

organism: Populus tremula x Populus alba

label_protocol: labelling Biotin indirect, amplification=yes, ssDNA 5.5 ug.

growth_protocol: stem - Acclimation on 03/14/11, transfer to hydroponic culture chamber on 05/24/11 growth media: NaCl 85µM; MgSO4 550µM; (NH4)2SO4 275µM; Ca(NO3)2 730µM; KNO3 1mM; K2HPO4 550µM; KH2PO4 550µM; (NH4)2MoO4 0.102nM; MnSO4 0.722nM; ZnSO4 0.612nM; H3BO3 1.812nM; CuSO4 0.246nM; 1/10000 masquolate ® FeA 2% - 16h/8h (day/night) - 22°C/18°C (day/night)

treatment_protocol: Name:TWO BENDING 30 M - environmental treatment - abiotic stress,stem bending:. HARVEST 30 MINUTES AFTER BEND

tag: treatment

value: TWO BENDING

tag: harvest protocol

value: HARVEST 30 MINUTES AFTER BEND


title: Healthy_Endometrium_gestation_day_20, biological replicate 3

description:
Pig 11 Healthy Endometrium
Experimental replicate 3 of 3 (Healthy Endometrium).

source_name: Endometrium associated with a healthy gestation day 20 conceptus, from sow #11


title: S.coelicolor germination_time 0 h_replicate 2


title: Arresting_Trophoblast_gestation_day_20, biological replicate 2

description:
Pig 12 Arresting Trophoblast
Experimental replicate 2 of 3 (Arresting Trophoblast).

source_name: Trophoblast associated with an arresting gestation day 20 conceptus (~50% smaller, and less vascularization than littermates), from sow #12


title: Healthy_Endometrium_gestation_day_20, biological replicate 2

description:
Pig 12 Healthy Endometrium
Experimental replicate 2 of 3 (Healthy Endometrium).

source_name: Endometrium associated with a healthy gestation day 20 conceptus, from sow #12


title: Vector rep1

description: Gene expression data from H1299 infected with an empty vector


title: S.coelicolor germination_time dormant_replicate 3

source_name: S.coelicolor germination time dormant


title: S.coelicolor germination_time 0 h_replicate 1

source_name: proportional mixture of RNA samples from all time points


title: Arresting_Endometrium_gestation_day_20, biological replicate 2

description:
Pig 12 Arresting Endometrium
Experimental replicate 2 of 3 (Arresting Endometrium).

source_name: Endometrium associated with an arresting gestation day 20 conceptus (~50% smaller, and less vascularization than littermates), from sow #12


title: PC1440_2 with array type poplar from Affymetrix

description:
populus tremula x populus alba  - harvest date: 20-05-11
genes regulated by PtaZFP2 in absence of mechanical stress - genes regulated by PtaZFP2 after one bending.Species: Populus tremula x Populus alba

growth_protocol: stem - - growth media: NaCl 85µM; MgSO4 550µM; (NH4)2SO4 275µM; Ca(NO3)2 730µM; KNO3 1mM; K2HPO4 550µM; KH2PO4 550µM; (NH4)2MoO4 0.102nM; MnSO4 0.722nM; ZnSO4 0.612nM; H3BO3 1.812nM; CuSO4 0.246nM; 1/10000 masquolate ® FeA 2% - 16h/8h (day/night) - 22°C/18°C (day/night)

extract_protocol: Pool of extract, PC1440A_2:125ng, PC1440B_2:125ng.

treatment_protocol: no treatment

value: no treatment


title: P1F4320_3 with array type poplar from Affymetrix

description:
populus tremula x populus alba  - harvest date: 15-06-11
genes regulated by PtaZFP2 in absence of mechanical stress - genes regulated by PtaZFP2 after one bending.Species: Populus tremula x Populus alba

growth_protocol: stem - growth media: NaCl 85µM; MgSO4 550µM; (NH4)2SO4 275µM; Ca(NO3)2 730µM; KNO3 1mM; K2HPO4 550µM; KH2PO4 550µM; (NH4)2MoO4 0.102nM; MnSO4 0.722nM; ZnSO4 0.612nM; H3BO3 1.812nM; CuSO4 0.246nM; 1/10000 masquolate ® FeA 2% - 16h/8h (day/night) - 22°C/18°C (day/night)

treatment_protocol: Name:ONE BENDING 24 HOURS  - environmental treatment - abiotic stress,stem bending:. HARVEST 24 HOURS AFTER BENDName: - environmental treatment - abiotic stress,stem bending:. HARVEST 72 HOURS AFTER BEND

value: ONE BENDING

value: HARVEST 24 HOURS AFTER BEND


title: uninfected control at 6 h PI

city: Bangalore

first: Genotypic

last: technology

state: Karnataka

address: 259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE

country: India

institute: Genotypic Technology

description: Gene expression of uninfected AD 293 cells at 6 h

hyb_protocol: The labeled cRNA samples were hybridized on to a Custom Whole Human Genome 4x44k designed by Genotypic Technology Private Limited. (AMADID: 025085). 1650ng of cy3 labeled samples were fragmented and hybridized.  Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5188-5242). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327)

scan_protocol: Scanned on an Agilent G2505C scanner and Images were quantified using Agilent Feature Extraction Software (version 10.7)

data_processing: The normalization was done using GeneSpring GX 11.5 Software. Recommended 75th Percentile Shift Normalization.

source_name: uninfected AD 293 cells  at 6 h

label_protocol: The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). Five hundred nanograms each of the Control and test samples were incubated with reverse transcription mix at 40°C and  converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up and quality assessed for yields and specific activity.

growth_protocol: The AD293 cells were grown in 35 mm2 tissue culture petri dish (corning) in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and antibiotics.

extract_protocol: The cells were harvested at indicated time points in RNA lysis buffer from PureLink RNA mini kit (Invitrogen). The total RNA was purified as per manufacturer’s protocol.

treatment_protocol: Confluent monolayer of AD293 cells in petridish was infected with 105 tissue culture infective dose 50% per mL (TCID50/ mL) of virus and incubated one hour at 37 0C with 5% CO2 for virus adsorption. After adsorption the cells were washed with DMEM medium and incubated with DMEM with 1% FBS.

value: Kidney

tag: transfected agent


title: P1F1440_3 with array type poplar from Affymetrix

treatment_protocol: Name:ONE BENDING 24 HOURS  - environmental treatment - abiotic stress,stem bending:. HARVEST 24 HOURS AFTER BEND


title: PC30_3 with array type poplar from Affymetrix

extract_protocol: Pool of extract, PC30A_3:125ng, PC30B_3:125ng.


title: P2F30_3 with array type poplar from Affymetrix

extract_protocol: Pool of extract, P2F30A_3:125ng, P2F30B_3:125ng.


title: S.coelicolor germination_time dormant_replicate 2


title: S.coelicolor germination_time dormant_replicate 1


title: Arresting_Trophoblast_gestation_day_20, biological replicate 1

description:
Pig 14 Arresting Trophoblast
Experimental replicate 1 of 3 (Arresting Trophoblast).

source_name: Trophoblast associated with an arresting gestation day 20 conceptus (~50% smaller, and less vascularization than littermates), from sow #14


title: P2F30_2 with array type poplar from Affymetrix

extract_protocol: Pool of extract, P2F30A_2:125ng, P2F30B_2:125ng.


title: Healthy_Trophoblast_gestation_day_20, biological replicate 1

description:
Pig 14 Healthy Trophoblast
Experimental replicate 1 of 3 (Healthy Trophoblast).

source_name: Trophoblast associated with a healthy gestation day 20 conceptus, from sow #14


title: Arresting_Endometrium_gestation_day_20, biological replicate 1

description:
Pig 14 Arresting Endometrium
Experimental replicate 1 of 3 (Arresting Endometrium).

source_name: Endometrium associated with an arresting gestation day 20 conceptus (~50% smaller, and less vascularization than littermates), from sow #14


title: ADN at 3 h PI

description: Gene expression of AD 293 cells with recombinant adeno virus expressing Chandipura virus N protein at 3 h PI

value: sheared adenovirus type 5 expressing Chandipura virus N protein DNA


title: Healthy_Endometrium_gestation_day_20, biological replicate 1

description:
Pig 14 Healthy Endometrium
Experimental replicate 1 of 3 (Healthy Endometrium).

source_name: Endometrium associated with a healthy gestation day 20 conceptus, from sow #14

value: Yorkshire


title: Recombinant adeno virus-6 h PI

description: Gene expression of AD 293 cells infected with recombinant adeno virus at 6 h PI

value: sheared adenovirus type 5 DNA


title: ADN at 6 h PI

description: Gene expression of AD 293 cells with recombinant adeno virus expressing Chandipura virus N protein at 6 h PI


title: P1F120_3 with array type poplar from Affymetrix

extract_protocol: Pool of extract, P1F120A_3:125ng, P1F120B_3:125ng.

treatment_protocol: Name:ONE BENDING 2 HOURS - environmental treatment - abiotic stress,stem bending:. HARVEST 2 HOURS AFTER BEND

value: HARVEST 2 HOURS AFTER BEND


title: P1F120_2 with array type poplar from Affymetrix

extract_protocol: Pool of extract, P1F120A_2:125ng, P1F120B_2:125ng.


title: gaiGR, 4h DEX-treated, biological replicate 3

city: Gent

first: Hannes

last: Claeys

address: Technologiepark 927

department: Plant Systems Biology

country: Belgium

institute: VIB-UGent

hyb_protocol: Standard Affymetrix protocol

scan_protocol: Standard Affymetrix protocol

data_processing: Data was processed with Robust Multichip Average (RMA) (background correction, normalization, and summarization) as implemented in BioConductor (Irizarry et al., 2003a; Irizarry et al., 2003b; Gentleman et al., 2004).

label_protocol: Standard Affymetrix protocol

growth_protocol: plant were grown on MS medium under 16 h light/ 8 h dark cycle at 21°C

extract_protocol: RNeasy plant mini kit (Qiagen), according to manufacturer protocol

treatment_protocol: Plant were transferred to MS medium containing 5 uM dexamethason or DMSO (mock) for 1 to 4 hours

tag: ecotype

value: leaf 3

tag: age

value: 5uM Dexamethasone

tag: genotype

tag: time

value: 4 h


title: gaiGR, 4h DEX-treated, biological replicate 2


title: WT_NT_rep1

city: Athens

first: Chung-Jui

last: Tsai

state: GA

address: 120 Green Street

department: School of Forestry and Natural Resources

institute: University of Georgia

description:
expression data from WT at NT
WTNT1

hyb_protocol: Agilent standard protocol

scan_protocol: Slides were scanned on an Agilent DNA Microarray Scanner at 3 µm resolution

data_processing: The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters to obtain background subtracted Processed Signal intensities(gProcessedSignal). Percentile Shift (75th) method was applied for normalization with Agilent's GeneSpring GX software

label_protocol: Agilent standard protocol

growth_protocol: Vegetatively propagated plants were randomly distributed to two identical chambers and grown at NT (27°C/17°C, day/night) to a height of ~120 cm.

extract_protocol: CTAB protocol

treatment_protocol: Plants in one chamber were exposed to HT (35°C/25°C, day/night)) for one week, while those in the other chamber remained at NT (27°C/17°C, day/night).  Leaf samples were harvested for RNA extraction.

value: wild type

tag: temperature

value: normal (27°C)

value: young source leaves at leaf plastochron index (LPI) 5


title: WT_NT_rep2

description:
expression data from WT at NT
WTNT2


title: gaiGR, 4h DEX-treated, biological replicate 1


title: PC4320_1 with array type poplar from Affymetrix

extract_protocol: Pool of extract, PC4320A_1:125ng, PC4320B_1:125ng.


title: N31_NT_rep1

description:
expression data from transgenic line N31 at NT
N31NT1

value: transgenic Populus expressing NahG

tag: transgenic line

value: NahG line N31


title: WT_HT_rep1

description:
expression data from WT at HT
WTHT1

tag: genotype/variation

value: high (35°C)


title: P1F4320_2 with array type poplar from Affymetrix

treatment_protocol: Name:ONE BENDING 72 HOURS  - environmental treatment - abiotic stress,stem bending:. HARVEST 72 HOURS AFTER BEND

value: HARVEST 72 HOURS AFTER BEND


title: gaiGR, 4h mock-treated, biological replicate 3

tag: tissue

value: mock


title: P1F30_2 with array type poplar from Affymetrix

extract_protocol: Pool of extract, P1F30A_2:125ng, P1F30B_2:125ng.

treatment_protocol: Name:ONE BENDING 30M - environmental treatment - abiotic stress,stem bending:. HARVEST 30 MINUTES AFTER BEND

value: ONE BENDING

value: HARVEST 30 MINUTES AFTER BEND


title: gaiGR, 4h mock-treated, biological replicate 2


title: gaiGR, 4h mock-treated, biological replicate 1


title: gaiGR, 1h DEX-treated, biological replicate 3

value: 1 h


title: 12-GMP_Tet2_Ezh2_DKO_RAEB

city: Chiba

first: ATSUSHI

last: IWAMA

address: 1-8-1 Inohana, Chuo-ku

department: Department of Cellular and Molecular Medicine

country: Japan

institute: Graduate School of Medicine, Chiba University

description: Gene expression data of GMP from Tet2KD/KDEzh2-/- mice that developed refractory anemia with excess of blasts (RAEB)

hyb_protocol: 1,650 ng of the labeled cRNA was hybridized to Agilent Whole Mouse Genome 4x44k array Ver.2, Design ID: 028005. The process of hybridization and washing was performed using a Hi-RPM Gene Expression Hybridization Kit (Large) (Agilent Technologies) and a Gene Expression Wash Pack  (Agilent Technologies) respectively.

scan_protocol: GeneChips were scanned using a DNA microarray scanner (Agilent Technologies)

data_processing: standard Agilent protocol

organism: Mus musculus

source_name: Tet2KD/KDEzh2-/-RAEB GMP from BM

label_protocol: 250 ng of total RNA was mixed with spike-in controls using an Agilent One Color Spike Mix Kit (Agilent Technologies), amplified and labeled with Cyanine 3 using a Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions, which generated single-color labeled cRNA.

tag: strain

value: GMP

tag: cells purified from

value: BM


title: 11-GMP_Tet2_Ezh2_DKO_MDS/MPN

description: Gene expression data of GMP from Tet2KD/KDEzh2-/- mice that developed myelodysplastic/myeloproliferative neoplasm (MDS/MPN)

source_name: Tet2KD/KDEzh2-/- MDS/MPN GMP from BM

value: Tet2KD/KDEzh2-/- MDS/MPN


title: PC4320_3 with array type poplar from Affymetrix

description:
populus tremula x populus alba  - harvest date: 18-04-11
genes regulated by PtaZFP2 in absence of mechanical stress - genes regulated by PtaZFP2 after one bending.Species: Populus tremula x Populus alba

value: HARVEST 2 HOURS AFTER BEND


title: WT_HT_rep2

description:
expression data from WT at HT
WTHT2


title: P1F30_3 with array type poplar from Affymetrix

extract_protocol: Pool of extract, P1F30A_3:125ng, P1F30B_3:125ng.


title: 10-GMP_Tet2_Ezh2_DKO

description: Gene expression data of GMP from Tet2KD/KDEzh2-/- BM

source_name: Tet2KD/KDEzh2-/- GMP from BM


title: P1F1440_2 with array type poplar from Affymetrix

value: HARVEST 24 HOURS AFTER BEND


title: PC120_2 with array type poplar from Affymetrix

extract_protocol: Pool of extract, PC120A_2:125ng, PC120B_2:125ng.

value: no treatment


title: N31_HT_rep1

description:
expression data from transgenic line N31 at HT
N31HT1


title: gaiGR, 1h DEX-treated, biological replicate 2

value: leaf 3

value: 5uM Dexamethasone


title: PC1440_3 with array type poplar from Affymetrix

extract_protocol: Pool of extract, PC1440A_3:125ng, PC1440B_3:125ng.


title: 9-GMP_Ezh2_SKO

description: Gene expression data of GMP from Ezh2-/- BM

source_name: Ezh2-/-GMP from BM

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


title: N31_NT_rep2

description:
expression data from transgenic line N31 at NT
N31NT2

value: normal (27°C)

value: young source leaves at leaf plastochron index (LPI) 5


title: gaiGR, 1h DEX-treated, biological replicate 1


title: PC4320_2 with array type poplar from Affymetrix

description:
populus tremula x populus alba  - harvest date: 20-05-11
genes regulated by PtaZFP2 in absence of mechanical stress - genes regulated by PtaZFP2 after one bending.Species: Populus tremula x Populus alba

extract_protocol: Pool of extract, PC4320A_2:125ng, PC4320B_2:125ng.


title: gaiGR, 1h mock-treated, biological replicate 3


title: PC1440_1 with array type poplar from Affymetrix

extract_protocol: Pool of extract, PC1440A_1:125ng, PC1440B_1:125ng.


title: 8-GMP_Tet2_SKO

description: Gene expression data of GMP from Tet2KD/KD BM

source_name: Tet2KD/KD GMP from BM


title: P1F1440_1 with array type poplar from Affymetrix


title: PC120_1 with array type poplar from Affymetrix

extract_protocol: Pool of extract, PC120A_1:125ng, PC120B_1:125ng.


title: I6_HT_rep2

description:
expression data from transgenic line I6 at HT
I6HT2

value: transgenic Populus expressing cytosolic Irp9

value: Irp9 line I6


title: 7-GMP_WT

description: Gene expression data of GMP from wild-type BM

source_name: Wild-type GMP from BM

value: Wild-type


title: gaiGR, 1h mock-treated, biological replicate 1

value: mock


title: I6_NT_rep2

description:
expression data from transgenic line I6 at NT
I6NT2


title: 5-LSK_Tet2_Ezh2_DKO_MDS/MPN

description: Gene expression data of LSK from Tet2KD/KDEzh2-/- mice that developed myelodysplastic/myeloproliferative neoplasm (MDS/MPN)

source_name: Tet2KD/KDEzh2-/- MDS/MPN LSK from BM

value: LSK


title: F10_NT_rep1

description:
expression data from transgenic line F10 at NT
F10NT1

value: transgenic Populus expressing plastidic Irp9

value: FD-Irp9 line F10


title: F10_NT_rep2

description:
expression data from transgenic line F10 at NT
F10NT2


title: 2-LSK_Tet2_SKO

description: Gene expression data of LSK from Tet2KD/KD BM

source_name: Tet2KD/KD LSK from BM


title: 6-LSK_Tet2_Ezh2_DKO_RAEB

description: Gene expression data of LSK from Tet2KD/KDEzh2-/- mice that developed refractory anemia with excess of blasts (RAEB)

source_name: Tet2KD/KDEzh2-/-RAEB LSK from BM


title: I6_HT_rep1

description:
expression data from transgenic line I6 at HT
I6HT1

value: high (35°C)


title: 4-LSK_Tet2_Ezh2_DKO

description: Gene expression data of LSK from Tet2KD/KDEzh2-/- BM

source_name: Tet2KD/KDEzh2-/- LSK from BM

value: BM


title: PC30_2 with array type poplar from Affymetrix

extract_protocol: Pool of extract, PC30A_2:125ng, PC30B_2:125ng.


title: PC120_3 with array type poplar from Affymetrix

description:
populus tremula x populus alba  - harvest date: 15-06-11
genes regulated by PtaZFP2 in absence of mechanical stress - genes regulated by PtaZFP2 after one bending.Species: Populus tremula x Populus alba

extract_protocol: Pool of extract, PC120A_3:125ng, PC120B_3:125ng.


title: 1-LSK_WT

description: Gene expression data of LSK from wild-type BM

source_name: Wild-type LSK from BM


title: F10_HT_rep2

description:
expression data from transgenic line F10 at HT
F10HT2


title: 3-LSK_Ezh2_SKO

description: Gene expression data of LSK from Ezh2-/- BM

source_name: Ezh2-/- LSK from BM


title: F10_HT_rep1

description:
expression data from transgenic line F10 at HT
F10HT1


title: P1F4320_1 with array type poplar from Affymetrix

description:
populus tremula x populus alba  - harvest date: 18-04-11
genes regulated by PtaZFP2 in absence of mechanical stress - genes regulated by PtaZFP2 after one bending.Species: Populus tremula x Populus alba

value: ONE BENDING

value: HARVEST 72 HOURS AFTER BEND


title: P1F30_1 with array type poplar from Affymetrix

extract_protocol: Pool of extract, P1F30A_1:125ng, P1F30B_1:125ng.

value: HARVEST 30 MINUTES AFTER BEND


title: nucleus accumbens_saline_treat_miRNA

city: Elmhurst

first: Chunxiang

last: Zhang

state: United States

address: 438 E May St

department: Pharmacology

institute: Rush University Medical Center

description: control group

hyb_protocol: Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http:/miRBase.org) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.

data_processing: Background is determined using a regression-based background mapping method. The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots. Raw data matrix is then subtracted by the background matrix. Normalization is carried out using a LOWESS (Locally-weighted Regression) method on the background-subtracted data. The normalization is to remove system related variations, such as sample amount variations, different labeling dyes, and signal gain differences of scanners so that biological variations can be faithfully revealed [B. M. Bolstad, R. A. Irizarry, M. Astrandand T. P. Speed, (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias, Bioinformatics, 19 (2), 185-193]. A transcript to be listed as detectable must meets at least two conditions: signal intensity higher than 3×(background standard deviation) and spot CV < 0.5. CV is calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level. t-Test is performed between “control” and “test” sample groups. T-values are calculated for each miRNA, and p-values are computed from the theoretical t-distribution. miRNAs with p-values below a critical p-value (typically 0.01) are selected for cluster analysis. The clustering is done using hierarchical method and is performed with average linkage and Euclidean distance metric. All data processes, except clustering plot, are carried out using in-house developed computer programs. The clustering plot is generated using TIGR MeV (Multiple Experimental Viewer) software from The Institute for Genomic Research.

organism: Rattus norvegicus

source_name: nucleus accumbens from saline treated rats

label_protocol: Microarray assay was performed using a service provider (LC Sciences). The assay started from 4 to 8 µg total RNA sample, which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent Cy3 dye staining.

growth_protocol: Male Sprague-Dawley rats (weight 150-180g at the start of the experiments) were used for this experiment. All animals were housed in separate cages in 12-h light and 12-h dark periods with free access to water and food.

extract_protocol: miRNAs were isolated from the NAc of rats using the mirVana miRNA isolation kit (Ambion, Inc.). miRNA expression profiling was done by miRNA microarray analysis using a chip containing 300 rat mature miRNAs (Chip ID miRRat 9.2 version; LC Sciences).

treatment_protocol: The animals were divided into two into two treatment groups: vehicle (500 μl saline, ip bid) or alcohol (1 g/kg, ip bid). Seven days later, the animals were sacrificed and their nucleus accumbens (NAc) were isolated for miRNA microarray analysis.The rats were sacrificed by decapitation at the end of experiments.  Rat brains were rapidly isolated and the frozen brain sections included the core and shell subdivisions of the NAc were cut as described previously

value: Sprague-Dawley

tag: gender

value: male

value: adult

tag: weight

tag: treated with

value: vehicle (500 ul saline, ip bid)

value: brain nucleus accumbens (NAc)


title: nucleus accumbens_alcohol_treat_miRNA

description: alcohol group

source_name: nucleus accumbens from alcohol treated rats

value: alcohol (1 g/kg, ip bid)


title: MORKO-LPS+Morphine

city: Nanjing

first: Yan

last: Zeng

state: Jiangsu

address: XuanWu District, No.1 WeiGang

country: China

institute: Nanjing Agricultural University

hyb_protocol: A mixture of labeled RNA and DNA was hybridized to a miRNA microarray (Thomson et al), inside a MAUI mixer in a MAUI hybridization system (BioMicro® System, Inc, Salt Lake City, UT, USA).     Thomson, J.M., Parker, J., Perou, C.M., and S.M. Hammond. 2004. A custom microarray platform for analysis of microRNA gene expression. Nat. Methods 1: 47-53. Epub 2004 Sep 29.

scan_protocol: Slides were scanned with a ScanArray 5000 machine (Perkin Elmer®, Waltham, MA, USA).

data_processing: BlueFuse (BlueGenome, Cambridge, UK) was used to quantify pixel intensities.  GeneSpring GX 7.3.1 (Agilent Technologies) was used for data normalization.

source_name: primary cells

label_protocol: Total RNA was ligated to a synthetic linker, 5’-pCU-DY547-3’.  Reference DNA oligonucleotides (Invitrogen) complementary to a subset of mammalian miRNAs were combined and labeled with a ULYSIS® Alexa Fluor® 647 kit.

growth_protocol: Macrophages harvested from mice (primary source)

extract_protocol: Trizol® reagent (Invitrogen) was used to extract total RNA from the cells.

treatment_protocol: All treatments done in vivo. Morphine implanted subcuteniously, LPS administered as intraperitoneal injection.

value: MORKO

tag: agent

value: LPS+Morphine


title: MORKO-LPS

value: LPS


title: WT-LPS

value: WT


title: WT-LPS+Morphine


title: F52_NT_rep1

description:
expression data from transgenic line F52 at NT
F52NT1

value: transgenic Populus expressing plastidic Irp9

value: FD-Irp9 line F52

value: normal (27°C)

value: young source leaves at leaf plastochron index (LPI) 5


title: F52_HT_rep2

description:
expression data from transgenic line F52 at HT
F52HT2

value: high (35°C)


title: F52_NT_rep2

description:
expression data from transgenic line F52 at NT
F52NT2


title: WT-control

value: control


title: PC30_1 with array type poplar from Affymetrix

extract_protocol: Pool of extract, PC30A_1:125ng, PC30B_1:125ng.

tag: treatment

value: no treatment


title: I6_NT_rep1

description:
expression data from transgenic line I6 at NT
I6NT1

value: transgenic Populus expressing cytosolic Irp9

value: Irp9 line I6


title: N31_HT_rep2

description:
expression data from transgenic line N31 at HT
N31HT2

value: transgenic Populus expressing NahG

value: NahG line N31


title: F52_HT_rep1

description:
expression data from transgenic line F52 at HT
F52HT1


title: MORKO-control


title: plasma from healthy donor [97B]

city: GuangZhou

first: jinze

last: zheng

address: Dongfeng east road 651#

institute: Sun Yat-Sen University Cancer Center

description: MicroRNA expression profiles in plasma

hyb_protocol: The labeling and hybridization procedures were performed according to the protocols in the Agilent microRNA Microarray System.

scan_protocol: The microarray image information was converted into spot intensity values using Feature Extraction Software Rev. 9.5.3 (Agilent Technologies, Santa Clara, CA). The signal after background subtraction was exported directly into the GeneSpring GX10 software (Agilent Technologies, Santa Clara, CA). .

data_processing:
The raw signals obtained for single-color CY3 hybridization were normalized to the endogenous control mir-454.
A log transformation of base 10 was performed before data analysis

source_name: plasma from healthy donor

label_protocol: Total RNA (100 ng) derived from the plasma samples was labeled with Cy3.

extract_protocol: RNA from 400 μL of plasma was isolated using the mirVana PARIS microRNA Isolation kit according to the manufacturer’s instructions (Ambion, Austin, TX). Total RNA from cells was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The RNA concentration was quantified using a NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Waltham, MA). 1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

treatment_protocol: For the plasma preparation, peripheral blood (3 ml) was drawn into EDTA tubes. Within 30 min, the tubes were centrifuged at 820 g for 10 min. Then, 1-ml aliquots of the plasma were transferred to 1.5-ml tubes and centrifuged at 16,000 g for another 10 min to remove any remaining cellular debris. Subsequently, the supernatants were transferred to fresh tubes and immediately stored at –80°C.

value: Peripheral Blood

value: male

value: 46 years


title: plasma from healthy donor [92A]

description: MicroRNA expression profiles in plasma

value: female

value: 45 years


title: plasma from healthy donor [87B]

description: MicroRNA expression profiles in plasma


title: plasma from healthy donor [54A]

description: MicroRNA expression profiles in plasma

value: 37 years


title: plasma from healthy donor [81B]

description: MicroRNA expression profiles in plasma

value: 49 years


title: plasma from healthy donor [85B]

description: MicroRNA expression profiles in plasma

value: 39 years


title: plasma from healthy donor [49B]

description: MicroRNA expression profiles in plasma

value: 59 years


title: plasma from healthy donor [84A]

description: MicroRNA expression profiles in plasma

value: 52 years


title: P1F120_1 with array type poplar from Affymetrix

extract_protocol: Pool of extract, P1F120A_1:125ng, P1F120B_1:125ng.

value: ONE BENDING

tag: harvest protocol

value: HARVEST 2 HOURS AFTER BEND


title: plasma from healthy donor [18A]

description: MicroRNA expression profiles in plasma

value: 63 years


title: 14286629 - col0_BR1 vs N575260_BR1

first: Soubigou-Taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

institute: INRA

description: Study of the biosynthesis of NAD in Arabidopsis. Involvment of QPTgene using the knock out Salk mutant N575260.

hyb_protocol: N575260_BR1 Cy5 / col0_BR1 Cy3 : 30pmol.  CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

scan_protocol: GenePix Pro 3.0, Cy3:pmt voltage 532nm,480V,laser power 35%, Cy5:635nm,pmt voltage 480V,laser power 30%

data_processing: The raw data comprised the logarithm of median feature pixel intensity at wavelength 635 nm (red) and 532 nm (green). No background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, we excluded spots that were considered badly formed features by the experimenter (Flags=-100). Then we performed a global intensity-dependent normalization using the loess procedure (see Yang et al., 2002) to correct the dye bias. Finally, on each block, the log-ratio median is subtracted from each value of the log-ratio of the block to correct a print-tip effect on each metablock.

label_protocol: labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug.  5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).

growth_protocol: rosette - Media=Soil hygrometry=65% Temperature=20°C Light=8 hours - 100-150 µmol quanta m-2 s-1

value: columbia

value: mutant (col0)

tag: dev.stage (boyes et al. plant cell 2001)


title: 14286473 - N575260_BR2 vs col0_BR2

hyb_protocol: col0_BR2 Cy5 / N575260_BR2 Cy3 : 30pmol.  CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

tag: ecotype


title: plasma from healthy donor [22B]

description: MicroRNA expression profiles in plasma

tag: age

value: 56 years


title: 14286397 - WT + MTX leaves Repet2 vs WT leaves Repet2

description: Folates and 1C metabolism

hyb_protocol: WT leaves Repet2 Cy5 / WT + MTX leaves Repet2 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

source_name: WT leaves Repet2

label_protocol: labelling Cy3 and Cy5 direct, amplification=yes, aRNA 5 ug. (Labelling_protocol.txt) Labelling protocol: 5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).

growth_protocol: caulin leaf - Media=Murashige and Skoog medium (pH 5.7) containing 1% (w/v) Suc, phytagar (0.5%–1%), and vitamins pyridoxine-HCl, nicotinic acid (0.5 mg/mL), and thiamine (1 mg/mL). Plates were incubated vertically in a growth room. For 5-Formyl-THF [(6S)-5-formyl-5,6,7,8-tetrahydrofolic acid, calcium salt; natural calciumfolinate; Schircks Laboratories] and Methotrexate [(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}phenyl)formamido]pentanedioic acid; Schircks Laboratories] supplementation, stock solutions were applied to the growth medium to achieve the desired working concentration. hygrometry- Temperature=19ºCNight-22ºCDay Light=120-140 mmol photons m2/s

extract_protocol: WT leaves Repet2:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.

value: wild type

value: leaves

source_name: WT + MTX leaves Repet2

extract_protocol: WT + MTX leaves Repet2:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.

treatment_protocol: Name:Methotrexate supplemented - compound based treatment - compound addition,methotrexate:quantity 5nM time 17day . Arabidopsis thaliana were seeded after cold treatment (3 days) and grown for 17 days in MS media with different treatments in growth room. After this time shoots and roots were collected and separated with a razor blade, flash frozen with liquid nitrogen and stored at -80ºC for further RNA extraction.

value: MTX


title: 14286631 - N575260_BR1 vs col0_BR1

hyb_protocol: col0_BR1 Cy5 / N575260_BR1 Cy3 : 30pmol.  CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 14286472 - col0_BR2 vs N575260_BR2

hyb_protocol: N575260_BR2 Cy5 / col0_BR2 Cy3 : 30pmol.  CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 14286395 - WT + MTX leaves Repet1 vs WT leaves Repet1

description: Folates and 1C metabolism

hyb_protocol: WT leaves Repet1 Cy5 / WT + MTX leaves Repet1 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

source_name: WT leaves Repet1

extract_protocol: WT leaves Repet1:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.

source_name: WT + MTX leaves Repet1

extract_protocol: WT + MTX leaves Repet1:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.


title: 14286396 - WT leaves Repet2 vs WT + MTX leaves Repet2

description: Folates and 1C metabolism

hyb_protocol: WT + MTX leaves Repet2 Cy5 / WT leaves Repet2 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: plasma from healthy donor [122B]

description: MicroRNA expression profiles in plasma

value: Peripheral Blood

value: male

value: 36 years


title: plasma from healthy donor [124B]

description: MicroRNA expression profiles in plasma


title: 14286392 - WT roots Repet3 vs WT + MTX roots Repet3

description: Folates and 1C metabolism

hyb_protocol: WT + MTX roots Repet3 Cy5 / WT roots Repet3 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.

source_name: WT + MTX roots Repet3

growth_protocol: roots - Media=Murashige and Skoog medium (pH 5.7) containing 1% (w/v) Suc, phytagar (0.5%–1%), and vitamins pyridoxine-HCl, nicotinic acid (0.5 mg/mL), and thiamine (1 mg/mL). Plates were incubated vertically in a growth room. For 5-Formyl-THF [(6S)-5-formyl-5,6,7,8-tetrahydrofolic acid, calcium salt; natural calciumfolinate; Schircks Laboratories] and Methotrexate [(2S)-2-[(4-{[(2,4-diaminopteridin-6-yl)methyl](methyl)amino}phenyl)formamido]pentanedioic acid; Schircks Laboratories] supplementation, stock solutions were applied to the growth medium to achieve the desired working concentration. hygrometry- Temperature=19ºCNight-22ºCDay Light=120-140 mmol photons m2/s

extract_protocol: WT + MTX roots Repet3:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.

value: roots

source_name: WT roots Repet3

extract_protocol: WT roots Repet3:10ug. (Amplification_Ambion.txt) Amplification des ARN messagers (Kit AMBION MessageAmp aRNA Amplification #1750) Le kit se compose de 3 parties : réactifs enzymatiques à -20°C, tampons de purification à 4°C et colonnes à température ambiante. Si les concentrations en ARN sont inférieures à 180 ng/µl, prévoir une précipitation. Transcription inverse A partir d'ARN totaux purifiés et quantifiés au Ribogreen : ARN tot (1µg)+ eau 5.5 µl T7-oligo-dT   0.5 µl Incuber dans le bloc chauffant à 65°C 10min, centrifuger brièvement , puis mettre sur glace. Préparer le Mix (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace :   10X First Strand Buffer :  1µl Ribonuclease Inhibitor :  0.5µl dNTP Mix :    2µl Reverse Transcriptase  0.5µl Ajouter 4 µl de ce mix dans les 6µl d'ARN, agiter légèrement et centrifuger brièvement. Volume final = 10 µl Incuber 2h dans un bain-marie à 42°C Centrifuger brièvement , puis mettre sur glace Allumer le bain-Marie à 16°C dans la chambre froide. Faire un mix dans cet ordre (prévoir 5% en plus) en mettant l'enzyme à la dernière minute, sur glace: Eau RNase-Free  31.5µl 10X 2nd Strand Buffer 5µl dNTP Mix    2µl DNA Polymérase  1µl RNase H.     0.5µl Ajouter 40 µl de ce mix aux 10 µl précédents. Volume final = 50 µl. Mélanger par pipetage et centrifuger brièvement. Incuber 2h à 16°C Ajouter 50 µl d'eau DEPC. Possibilité de garder les échantillons après cette étape à -20°C en attendant la purification Purification des cDNA: cette étape doit se faire le plus tard possible Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. À 4 °C, le Binding Buffer peut avoir cristallisé, il faut bien le redissoudre avant utilisation en le chauffant brièvement au bain marie à 50°C. Equilibrer la colonne: déposer 50 µl de cDNA Binding Buffer sur la colonne cDNA Incuber 5 min à température ambiante. (Ne pas centrifuger) Ajouter 250µl de cDNA Binding Buffer dans les 100µl d'[ADN + eau] Mélanger par pipetage Déposer le tout sur la colonne équilibrée Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube 2 ml Lavage : déposer 500 µl de cDNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer colonne dans un tube de 1.5 ml Déposer au milieu de la colonne 5µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10000g à température ambiante Répéter l'élution dans le même tube avec 5µl d'eau RNase-free à 50°C et éteindre le bain-marie Transcription in vitro pour la synthèse d'aRNA(prévoir 5% en plus) : Préparer le mix suivant en mettant l'enzyme à la dernière minute, sur glace : 2µl ATP Soln (75mM) 2µl CTP Soln (75mM) 2µl GTP Soln (75mM) 2µl UTP Soln (75mM) 2µl T7 10X réaction Buffer Attention : il peut avoir cristallisé : bien le redissoudre avant utilisation 2µl T7 enzyme Mix Mélanger par pipetage, centrifugation brève Ajouter 12 µl de ce mix dans les 8 µl précédents. Volume final = 20 µl Incuber 14h dans l'étuve "bactério" à 37°C. Rajouter 1 µl de DNase I, mélanger par pipetage, centrifugation brève Incuber 30min dans le bloc chauffant à 37°C. Possibilité de conserver à -20°C avant la purification Ajouter 80 µl "Elution Solution" à l'éluat. Vf = 100µl. Purification des aRNA : Allumer un bain-Marie et le régler à 50 °C. Y mettre l'eau RNase-free à préchauffer. Ajouter aux 100 µl précédents :  350µl aRNA Binding Buffer  250µl EtOH 100%. Mélanger par pipetage et déposer l'échantillon sur le centre de la colonne aRNA Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et replacer la colonne dans le tube de 2 ml Lavage : déposer 650µl aRNA Wash Buffer sur la colonne (Vérifier que l'éthanol a bien été ajouté) Centrifuger 1 minute à 10 000g à température ambiante Jeter le liquide et centrifuger à nouveau 1 minute à 10 000g afin d'éliminer les traces d'éthanol Transférer la colonne dans un tube de 1.5 ml Elution : déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C Incuber 2 minutes à température ambiante et remettre l'eau à 50°C Centrifuger 1,5 minutes à 10 000g à température ambiante Répéter l'élution dans le même tube : Déposer au milieu de la colonne 50µl d'eau RNase-free à 50°C et éteindre le bain-marie Incuber 2 minutes à température ambiante Centrifuger 1,5 minutes à 10 000g à température ambiante Le volume final est entre 90 et 100 µl. Quantification : Préparer une dilution au 1/20 pour chaque échantillon et quantifier au ribogreen. Si la quantité finale est inférieure à 50 µg, vérifier la qualité sur gel (ou puce Bio-Analyser.) Si la concentration est inférieure à 625 ng/µl, prévoir une précipitation pour le marquage : Ajouter 10 µl de Na Acetate 3M, pH 5,2 et 250 µl d'Ethanol 96,2% Précipiter 1 heure à -20°C ou 30 minutes à -80°C Centrifuger 30 minutes 13 000 rpm à 4°C Vider le surnageant et laver le culot avec 500 µl d'éthanol à 70 % Centrifuger 5 minutes à 13 000 rpm à 4°C et pipeter le surnageant Laisser le culot sécher à l'air libre sur glace pendant 15 minutes Resuspendre avec de l'eau RNase-free à une concentration de 1 µg/µl.


title: plasma from healthy donor [249]

description: MicroRNA expression profiles in plasma

value: female

value: 54 years


title: 14286393 - WT + MTX roots Repet3 vs WT roots Repet3

description: Folates and 1C metabolism

hyb_protocol: WT roots Repet3 Cy5 / WT + MTX roots Repet3 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.


title: 14286394 - WT leaves Repet1 vs WT + MTX leaves Repet1

description: Folates and 1C metabolism

hyb_protocol: WT + MTX leaves Repet1 Cy5 / WT leaves Repet1 Cy3 : 30pmol. (Hybridization_Protocol.txt) Hybridization Protocol: CATMA slides (Corning Microarray Technology, CORNING) are pretreated in the prehybridisation solution (1 % BSA, 0.1 SDS, 5X SSC ) at 42°C for 60 min. They are dipped a couple of times in distilled water at room temperature, then in isopropanol and dried immediately by compressed nitrogen stream. Slides were placed in Corning hybridization chambers with a 25x60 lifterslip and 10ul of distilled water for each groove. The target was diluted to a final volume of 60 µL as follows 15µl of purified, labeled cDNA, 15 µl of 4X Hybridization Buffer ( 20X SDS, 0.4 % SDS), 30 µL formamide. The target mixture is heated for 3 min at 95°C, put on ice for 30 sec and centrifuged to remove dust for 1 min. The target mixture was put on the chip as quickly as possible. The microarray is sealed in a chamber and submerged in a 42°C water bath for approximately 16 h. The microarray is washed for 4 min in 1xSSC, 0.2% SDS (42°C); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC, 0.2% SDS (RT); 4 min in 0.1x SSC (RT); dipped a few times in distilled water and dried immediately by compressed nitrogen stream.