MGED informations
These guidelines are based on the document called Minimum Information About a Microarray Experiment – MIAME [1], developed by the Microarray Gene Expression Data society.

Sources for describe microarray experiments :
http://www.mged.org/Workgroups/MIAME/miame_1.1.html
http://www.mged.org/Workgroups/MIAME/miame_checklist.html
http://mged.sourceforge.net/ontologies/MGEDontology.php

Experiment : Validation by Francois and Philippe.
•	Type of experiment: for example, is it a comparison of normal vs. diseased tissue, a time course, or  is it designed to study the effects of a gene knock-out?
•	Experimental factors: the parameters or conditions tested, such as time, dose, or genetic variation.
•	The number of hybridizations performed in the experiment.
•	The type of reference used for the hybridizations, if any. 
•	Hybridization design: if applicable, a description of the comparisons made in each hybridization, whether to a standard reference sample, or between experimental samples. An accompanying diagram or table may be useful.
•	Quality control steps taken: for example, replicates or dye swaps.
•	URL of any supplemental websites or database accession numbers

Experimental design

Fields	Short description	Bernard-Pierrot I. et al, 2004	Validation status
 or 	
Type of experiment:	[MGED Ontology definition]	Comparison of normal vs transfected cells		
Experimental factor:	[MGED Ontology definition]	Transfection of FGFR2b		
Number of hybridizations:	[MGED Ontology definition]	8		
Hybridization design*:	[MGED Ontology definition]	Transfected vs non tranfected, with 4 replicates		
Quality control steps:	[MGED Ontology definition]	- technical_replicate
- within and between Nylon membrane replicates		
Experiment description:	A brief description of the experiment and its goal and a link to a publication if exists	The aim of this study is to investigate the effect of the fibroblast growth factor receptor 2b (FGFR2b) expression on human bladder cancer cells. Thus, a human tumour cell line T24 was transfecting with a vector containing the full-length FGFR2 cDNA under the control of the cytomegalovirus promoter. The vector without an insert was used as a control (for more details, see Ricol et al., 1999).		
URL of any supplemental websites:	[MGED Ontology definition]			
•	*An accompanying diagram or table may be useful.

Samples used, extract preparation and labeling: Validation by Francois and Andy.
•	The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: for example, gender, age, developmental stage, strain, or disease state.
•	Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques.
•	Protocol for preparing the hybridization extract: for example, the RNA  or DNA extraction and purification protocol.
•	Labeling protocol(s).
•	External controls (spikes).

Sample description :

Fields	Short description	Samples used in experiments	Validation status
 or 	
Name	a unique name or ID that describes your sample	T24N4	T24K10		
Organism	[MGED Ontology definition]	Homo sapiens neanderthalensis, NCBI_taxonomy, http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/		
Gender	[MGED Ontology definition]	Female (from ATCC)			
Sample provider		UMR 144 du CNRS, Institut Curie, Section Recherche
26 rue d’Ulm 75248 Paris Cedex, France
Francois.radvany@curie.fr		
Sample type	[MGED Ontology definition]	Cell		
Developmental stage	[MGED Ontology definition]	Adult		
Age	[MGED Ontology definition]	81 (from ATCC)		
Initial time point	[MGED Ontology definition]	Birth (to the age)		
Organism part	[MGED Ontology definition]	Bladder (from ATCC)		
Genetic modification	[MGED Ontology definition]		Expression of FGFR2b		
Individual identifier	[MGED Ontology definition]	HTB-4		
Individual genetic trait or genotype	[MGED Ontology definition]				
Disease state	[MGED Ontology definition]	Carcinoma		
Cell type or target cell type	[MGED Ontology definition]	Epithelial		
Cell line, animal strain or cultivar	[MGED Ontology definition]	HTB-4, ATCC, 
http://www.atcc.org/SearchCatalogs/CellBiology.cfm		
In vivo or in vitro treatment	[MGED Ontology definition]				
Additional information		Ricol et al., 1999. Oncogene 18, 7234-43		

Sample growth conditions protocols :
A description of the isolated environment used to grow organisms (samples) or parts of the organism (e.g. tissues, cells) [MGED Ontology Definition].

Fields	Bernard-Pierrot I. et al, 2004	Validation status
 or 	
Temperature :	37°C and 5% CO2		
Medium :	DMEM/F12 (Dubelco’s modified Eagle’s medium ) with
2 mM glutamine,
100 units/ml penicillin,
100 g/ml streptomycin,
10 mM HEPES,
50 nM hydrocortisone,
5 µg/ml apotransferin,
5 nM sodium selenium,
400 g/ml G418,
and supplemented by 10% heat-inactivated fetal calf serum (FCS).		
Full protocol description : (All the details)	T24N4 and T24K10 were cultured at 37°C and 5% CO2 T24 inDMEM/F12 (Dubelco’s modified Eagle’s medium ) containing 2 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 10 mM HEPES, 50 nM hydrocortisone, 5 µg/ml apotransferin and 5 nM sodium selenium, 400 g/ml G418 and supplemented by 10% heat-inactivated fetal calf serum (FCS). The cultured cells are replated twice a week.		

Sample separation technique protocol :

Fields	Bernard-Pierrot I. et al, 2004	Validation status
 or 	
Extracted nucleic acid 	total RNA		
Amplification :	????? (ex :RNA polymerase)		
Full protocol description : (All the details)	Total RNA prepared from both types of cells using RNeasy mini-kit as per manufacturer’s instructions.		

Labeling protocol 

Fields	Bernard-Pierrot I. et al, 2004	Validation status
 or 	
Amount of nucleic acid labeled 	10µg		
Label used :	33P		
Amplification :	?????		
Full protocol description : (All the details)	Reverse transcriptions
1-	In 1.5ml tube : heat 70°C x 5min an dRT x5min

10µg total RNA                     | ?
RNAse free water                   | ?
200 pmol oligo T16 VN (200 pmol/µl)| 1
-----------------------------------|----
final volume                       |11µl

2-	1st strand synthesis for each reaction :

oligo T primer total RNA from above |11
5x 1st strand buffer                | 4
0.1M DTT                            | 2
10mM dNTPs                          | 2
SuperScript II RT (200U/µl)         | 1
------------------------------------|----
final volume                        |20µl

3-	Heat reactions 42°C x 1h to synthesise 1st strand cDNA
4-	Add 1µl RNAse H and heat 37°C x 30 min to degrade template
5-	Purify cDNA using QIAquick PCR purification column as per manufacturer’s protocol (eluted 50µl). Dry the purified cDNA under vacuum.
6-	Dissolve dried cDNA in 32µl sterile water.

Radiolabelling of cDNA by Random priming
1-	Heat cDNA 70°C x 5min and to RT x 5min then add remaining reagents

1st strand cDNA                     |32
BSA (10mg/ml)                       | 5
OLB for dATP                        |10
33P-dATP [2500Ci/mmol, 10            | 2
Klenow (NEB 5U/µl)                  | 1
------------------------------------|----
final volume                        |50µl

2-	Heat 30min at 37°C to synthetise labelled probe
3-	Purify probe using ProbeQuant G-50 micro column as per manufacturer’s instructions.

Reagents
Rnase H (LifeTechnologies, 18021-014)
SuperScript II reverse transcriptase plus buffers (LifeTechnologies, 18064-014)
ProbeQuant G-50 Micro Columns (AP Biotech, 27-5335-01)
Klenow (5U/µl) (NEB, 210S)
QIAquick PCR Purification Kit (QIAGEN, 28104)
QIAGEN Rneasy mini kit (QIAGEN, 74103)
DNAse I (LifeTechnologies, 18047-019)		

Hybridization procedures and parameters: Validation by Andy.
•	The protocol and conditions used during hybridization, blocking and washing.
Fields	Bernard-Pierrot I. et al, 2004	Validation status
 or 	
Hybridization chamber type 	?????		
Quantity of the labelled extract used:	10µg of total RNA?????		
Duration
the time length of the hybridization	16h		
Hybridization volume 
the total volume of the hybridization	10ml		
Temperature 
the temperature of the hybridization	62°C		
Full protocol description (All the details)	•	Nylon membrane pre-treatement 
The nylon membranes, that are cross linked, are placed in a roller bottle with the following solutions
-	20 ml 0.5MNaOH/1.5%M NaCl and rotate x3min at RT
-	20 ml 0.5MTris/1.5%M NaCl and rotate x3min at RT
-	20 ml 2x SSC and rotate x3min at RT
-	decant final solution, invert and leave Nylon membrane in bottle ready for pre-hybridisation solution
•	Pre-hybridizations
The Nylon membranes were pre-hybridised at 62°C for 30min with 10mL hybridisation buffer (~60µl/cm2)in a 25cm Techne bottle.
•	Hybridizations
1-	The radio labelled probes were each mixed with 250µl human Cot I DNA to give a final concentration of 25ng/µl in the hybridization solution, denatured 5min x 95°C, and added directly to prehybridized buffer.
2-	The hybridization were done at 62°C for 16h with rotation.
•	Membrane Washing
1-	Each Nylon membrane was quickly rinsed once with ~100ml wah solution to remove excess probe and then washed twice with ~100ml wah solution at 62°C for 30min.
2-	The Nylon membranes were paced between clingfilm and exposed for 72h to Molecular Dynamics storage phosphor plates. Note- the exposure time varies with the age of the 33P isotope. Generally speaking fresh isotope only requires a 24h exposure to a storage phosphor screen. The storage phosphor plates were sanned in a Molecular Dynamics STORM instrument and the result *.GEL files loaded into ArrayVision software for quantitation of the hybridisation signals.

•	Reagents
Formamide (de-ionised) (SIGMA, F 9037)
NaH2PO4 (SIGMA, S 3139)
Na2HPO4 (SIGMA, S 3264)
human Cot I DNA 1mg/ml (LifeTechnologies, 15279-011)
•	Solutions
- Sodium phosphate buffer : NaH2PO4 (mw 120) 0.28M, Na2HPO4 (mw 142) 0.72M,dH2O
- Hybridisation buffer : 0.2M sodium phosphate buffer, 6.7% SDS, 15% formamide, 1% BSA, 1mM EDTA
- Wash solution : 40mM sodium phosphate buffer, 1% SDS		

Measurement data and specifications: Validation by Andy and Philippe
•	The quantitations based on the images. 
•	The set of quantitations from several arrays upon which the authors base their conclusions. While access to images of raw data is not required (although its value is unquestionable), authors should make every effort to provide the following: 
•	Type of scanning hardware and software used: this information is appropriate for a materials and methods section.
•	Type of image analysis software used: specifications should be stated in the materials and methods.
•	A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis.
•	The complete output of the image analysis before data selection and transformation (spot quantitation matrices).
•	Data selection and transformation procedures.
•	Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices).
Fields	Bernard-Pierrot I. et al, 2004	Validation status
 or 	
Scanner type 	Storm 860 PhosphorImager (Molecular Dynamics)		
Scanning software 	ImageQuant (Amersham Biosciences)?????		
Quantitation software 	Arrayvision 5.1, Imaging Research Inc.		
Quantitation type 	- VOL – MDC measurement (= pixel density multiplied by area). The volume gives the total number of counts (MDC = Molecular Dynamics Counts) within each circle that is placed over every spot on the array image.
- No background substraction is applied.
Note- The software ‘read’ the membrane into a left to right fashion, ie A1 to A24 then B1 to B24 and so on until H24.		
Spot quantitation matrices 	See the file ….. (raw data)		
Data selection and transformation procedures :	Description :

In the analysis, we consider each log gene expression measurement to be an additive sum of four terms: (i) gene effect, (ii) array effect nested in the cell line, (iii) cell line effect, (iv) interaction effect between gene and cell line, (v) error term. Systematic variation related to the experimental design array are considered as fixed effects, whereas the unknown quantities of interest (gene and gene-cell interaction) are considered as random effects. As the final term of interest is the gene-cell interaction term which reflects differential gene expression among the two cell lines for a defined gene, we calculate these gene-based statistics through a classical analysis of variance model. Thus, we modelize the marginal distribution of the gene-based statistics in a hierarchical Bayesian mixture formulation with an unknown number of components (Broët et al. 2002) and estimation of the model was implemented using stochastic algorithms. This adaptative statistical model allows for describing several levels of gene expression changes which correspond to the components of the mixture. In this model, the mean parameter for the unmodified component (which corresponds to the lack of gene-cell interaction) is set to zero (Broët et al. 2004). Posterior probabilities for the genes to belong to components related to different level of under or over expression were obtained together with the posterior distribution of the number of components. Based on this later distribution, we chose to focus on the largest plausible number of components for the mixture model. Finally, in order to avoid having to define a threshold posterior probabilities for choosing the set of genes to be explored, we propose to use a simple classification rule which assigns each gene to the component of the mixture to which it has the highest probability of belonging (Bayes allocation rule). We then list the genes that are attributed to the extreme components, the choice of extreme components being based on their estimated means. More details on the statistical procedures are given in the papers of Broët et al. (2002, 2004).

Steps :
1-	averaging over the duplicated features (arithmetic mean)
[moyenne des duplicats]
2-	log-transformation of the data
[log transformation des données]
3-	application of an additive model (gene effect, array effect, cell line effect and interaction gene-cell line effect, please see the article Broët et al, 2002)
[Modèle additif (effets gene, lame, lignée, interaction, cf article]
4-	Extraction of the parameter of interest : gene-cell line interaction (please see the article Broët et al, 2002)
[Extraction du paramètre d’intérêt (interaction gene*lignée)]
5-	Statistic analysis to quantify differential expression of gene-cell line interaction (between 2 cell lines)
[Calcul d’une statistique (différentiel d’interaction gène*lignée(entre les 2 lignées))]
6-	Modelisation of statistics marginal distribution following a mixture model (hierarchical Bayesian mixture)
[Modélisation de la distribution marginale des statistiques selon un modèle de mélange (Bayésien hierarchique)]
7-	To calculate the posterior probabilities (for each gene) of belonging to the non modified group
[Calcul de la probabilité a postériori (pour chaque gène) d’appartenance au groupe dit non modifié]		
Final gene expression data table(s)	See the file ….. (analysed data)		

Array Design : Validation by Andy and Severine
•	General array design, including the platform type (whether the array is a spotted glass array, an in situ synthesized array, etc.); surface and coating specifications (when known – often commercial suppliers do not provide this data); and the availability of the array (the name or make of commercially available arrays).

Fields	Short description	Bernard-Pierrot I. et al, 2004	Validation status
 or 	
Technology type	[MGED Ontology definition]	Spotted Nylon membrane
spotted_ds_DNA_features
(please go to the link, to know if it is ok because there is other type of technology type)		
Surface type 	[MGED Ontology definition]	?????		
Attachment type	[MGED Ontology definition]	?????		
Array design name:
version:		
?????
?????		
Array manufacturing protocol		The clone inserts from a number of public and proprietary cDNA librairies were amplified by PCR, checked by agarose gel electrophoresis, and the un-purified products printed onto 22.6cm * 22.6cm NytranSupercharge nylon membranes (Shleider + Scheull) in a pre-designated pattern. Each memebrane was cut into 3 sets of 2 fields (representing 96 well plates positioned end to end) which measured approximatively 7.53cm * 22.6cm, a cut across the bottom right hand corner is included for orientation.
Each field consists of 96 primary grids (A1-H12). The primary grids are comprised of duplicated individual elements (ie sopts of PCR product on the memebrane) from pre-selected 96 well plates containing un-purified PCR products.
There are 2 fields * 96 primary grids * 48 elements = 9216 sopts per memebrane ie 4608 PCR products spotted in duplicate (plate 49 in each case contains only blank wells).
The two fields shown in the diagram above have 7 * 7 spotting patterns for each primary grid :

The PCR products are UV cross linked to the nylon membrane by using a Startalinker 2400 (Startagene) on AUTO setting (to avoid spot smearing, “comet tails” and so on).		
•	
•	
•	
•	
•	For each feature (spot) on the array, its location on the array and the ID of its respective reporter (molecule present on each spot) should be given. 
•	For each reporter, its type (e.g., cDNA or oligonucleotide) should be given, along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database reference(s) and sequence (if available).
•	For non-commercial arrays, the following details should be provided:
o	The source of the reporter molecules: for example, the cDNA or oligo collection used, with references.
o	The method of reporter preparation.
o	The spotting protocols used, including the array substrate, the spotting buffer, and any post-printing processing, including cross-linking.
o	Any additional treatment performed prior to hybridization

cDNA array description file, example
Feature	Reporter	Composite sequence	
Coordinates on Array	Reporter ID (user defined) HGMP Ref	Biosequence Type	Clone ID
	DDBJ/
EMBL/
Genbank	Reporter Usage	Control Type	Comp. ID	Designation	Related Gene Symbol	Database
Entry
	
Meta
Col	Meta
Row	Col	Row											
1	1	1	1	370503	cDNA clone	IMAGE
32017	R17905	Experimental	_	C1	Gene	FNTA	LocusID2339	
1	1	2	1	370504	cDNA clone	IMAGE
2962831	BC005866	Experimental	_	C2	Gene	MLH1	LocusID 4292	
1	1	3	1	370505	Genomic clone	Cosmid
9H11	L40416	Control	Positive 	_	_	_	_	

With our data, example
Feature	Reporter	Composite sequence	
Coordinates on Array	Reporter ID (user defined) HGMP Ref	Biosequence Type	Clone ID
	DDBJ/
EMBL/
Genbank	Reporter Usage	Control Type	Comp. ID	Designation	Related Gene Symbol	Database
Entry
	
Meta
Col	Meta
Row	Spot num											
A	1	48b	4514	cDNA clone	???	???	Experimental	_	C1	Gene	LAR	????	
A	1	Blank	1	-	-	-	Control	Control_buffer 	_	_	_	_