The  MIAME Checklist
Experiment Design:
•	Type of experiment: Highly metastatic RMS cell lines vs. Poorly metastatic RMS cell lines   (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: Metastatic potential (the parameters or conditions tested, such as time, dose, or genetic variation.)
•	The number of hybridizations performed in the experiment : 27
•	The type of reference used for the hybridizations, if any: RMS772 cells
•	Hybridization design: Each hybridization employed a standard reference sample, namely the RNA of the RMS772 cell line. (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: We performed dye-reversal on 9 of the 18 unique samples, producing a total of 27 hybridizations. (for example, replicates or dye swaps.)
•	URL of any supplemental websites or database accession numbers

Samples used, extract preparation and labeling: 
•	The origin of the biological sample (for instance, name of the organism, the provider of the sample) and its characteristics: Highly and poorly metastatic RMS cell lines derived from neoplasms arising in HGF/SF transgenic ink4a/arf deficient mice, generated on an FVB - C57BL/6 background as previously described (Sharp et al., 2002).   (for example, gender, age, developmental stage, strain, or disease state.)
•	Manipulation of biological samples and protocols used: RMS cells were maintained in RIPM1640 medium including 10% FBS (Gibco).  All microarray experiments were performed with relatively early passage cells (passages 3-15).  Both murine wild-type Ezrin and dominant-negative Ezrin T567A were subcloned into the pEGFPN1 expression vector.  The mouse Six1 expression plasmid was a generous gift from Dr. Pascal Maire, INSERM.  Overexpression vectors were transfected into cell lines using lipofectamine plus (Invitrogen).  For all constructs at least two stable lines were made and tested for all assays.  (for example, growth conditions, treatments, separation techniques.)
•	Protocol for preparing the hybridization extract: Total RNA was extracted from highly and poorly metastatic RMS cell lines using Trizol according to the manufacturer’s recommendations (Life Technologies Inc, Rockville, MD).  After quantification by spectrophotometry and 1.2% agarose gel electrophoresis, total RNA was used for microarray experiments and/or Northern blot hybridization experiments.  Radiolabeled probes included mouse and human Six1, and human Erzin.  (for example, the RNA  or DNA extraction and purification protocol.)
•	Labeling protocol(s): Fluorescently-labeled cDNA, using either Cy3 or Cy5 (Amersham Pharmacia Biotech, NJ), was synthesized from 100-200 mg total RNA by oligo dT-primed polymerization using SuperScript II reverse transcriptase (Life Technologies, MD), as previously described (Khan et al., 1999b).
•	External controls (spikes).
Hybridization procedures and parameters:  
•	The protocol and conditions used during hybridization, blocking and washing.
Measurement data and specifications: 
•	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: 
o	Type of scanning hardware and software used: GenePix Scanner and acquisition software (GenPix, Inc.)
o	Type of image analysis software used: DeArray Software (Chen et al. 1997)
o	A description of the measurements produced by the image-analysis software and a description of which measurements were used in the analysis: “calRatio” was used in the analysis and “rQuality” was used for data filtering.
o	
Column Title	Description	Data Type	
CloneIndex	Index to the gipo file. 1 - first item in the GIPO file, 2 - second, …	Integer	
PenAt	Pen Position. It is always numbered from left-to-right, and top-to-bottom. (serpentine, start from top-left)	Integer	
RowAt	Within each sub-array (pen), row offset (start from 1 at top-most) to the spot of interest	Integer	
ColumnAt	Within each sub-array (pen), column offset (start from 1 at left-most) to the spot of interest	Integer	
SR_top	Bonding box location in Red channel image coordinates - (top, left, bottom, right) (SR -- Sample in Red channel)	Integer	
SR_left	 	Integer	
SR_bottom	 	Integer	
SR_right	 	Integer	
SR_T. Int.	Total Intensity - Sum of all pixel gray-levels within the target area. No data trimming is performed here.	Integer	
SR_Mean	Mean Intensity - Average of all pixel gray-leves within the target area, after trimming top and bottom 5%	float	
SR_S.Dev	Std. Dev. Of intensity - Std. Dev. Of signal intensity. Derived along with mean intensity	float	
SR_SNR	Signal-to-Noise Ratio. It is roughly the ratio of mean signal intensity to the local background std. Dev.	float	
SR_iQuality	The measurement quality. Summarized from Area, Background, Consistency, and Saturation. From 0 (bad)  to 1 (good)	float	
SR_bkMean	Mean background Intensity 	float	
SR_bkDev	Std. Dev. Of background intensity	float	
SG_top	Bonding box location in Red channel image coordinates - (top, left, bottom, right) (SG -- Sample in Green channel)	Integer	
SG_left	 	Integer	
SG_bottom	 	Integer	
SG_right	 	Integer	
SG_T. Int.	Total Intensity - Sum of all pixel gray-levels within the target area. No data trimming is performed here.	Integer	
SG_Mean	Mean Intensity - Average of all pixel gray-leves within the target area, after trimming top and bottom 5%	float	
SG_S.Dev	Std. Dev. Of intensity - Std. Dev. Of signal intensity. Derived along with mean intensity	float	
SG_SNR	Signal-to-Noise Ratio. It is roughly the ratio of mean signal intensity to the local background variation.	float	
SG_iQuality	The measurement quality. Summarized from Area, Background, Consistency, and Saturation. From 0 (bad)  to 1 (good)	float	
SG_bkMean	Mean background Intensity 	float	
SG_bkDev	Std. Dev. Of background intensity	float	
unionArea	Union Area - the detected target area. All calculations are based on this region	Integer	
propArea	Proportional area relatively to the mask size.	float	
calRatio	Calibrated ratio, (or normalized ratio)	float	
rQuality	Ratio measurement quality (including all intensity measurement quality, plus the signal-to-noise-ratio requirement).	float	
confLevel	Confidence Level, typically set at 99%	float	
lowerLimit	Lower Limit of confidence interval for cal. Ratio	float	
upperLimit	Upper Limit of confidence interval for cal. Ratio	float	
M	Magnitude parameter for channel compensation	float	
CV	Coefficient of variation of the intensity	float	
Flag	User flagged. 0 - deleted spot by user, 1 - good spot	Integer	
CloneTitle	Title of clone	String	
CloneID	Clone ID	String	
PlatePos	Plate location	String	
HKFlag	House Keeping flag	Integer	
GelBand	Number of bands	Integer	
Cytoband	Cytogenetics map	String	
geneMap	GeneMap-99 location	String	
o	
o	The complete output of the image analysis before data selection and transformation (spot quantitation matrices): Individual image analysis output files are in the folder named “Image Analysis Output”. 
o	Data selection and transformation procedures. The genes were filtered to include only those genes whose average “rQuality” from above table was > 0.5 across all 27 experiments according to previously published algorithms (Chen et al., 2002).  The remaining features are in this table:  “Yu_et_al_3467genes.txt”.   We then calculated a discriminative weight () for each gene calculated by  = dB/ (k1d1 + k2d2 +) where dB is the center-to-center distance (between cluster Euclidean distance), d is the average Euclidean distance among all sample-pairs, total of t1 and t2 sample pairs for cluster 1 (high) and 2 (low), respectively, and k1 = t1/(t1+t2), and k2 = t2 / (t1+t2) (Bittner et al., 2000).   is a small constant (0.1) to prevent a zero denominator.  Genes were then ranked on the basis of .  We next randomized the labels of the samples 1000 times and calculated both p and  as described (Ringner et al., 2002), and selected genes whose weight was >1.4 (p<0.001, <0.001), which resulted in the survival of 44 elements.
o	Final gene expression data table(s) used by the authors to make their conclusions after data selection and transformation (gene expression data matrices): The 44 differentially expressed genes can be found in this table: “Yu_et_al_44genes.txt”

Array Design:
•	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): In-house spotted glass cDNA array, 
•	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.: See table “cDNA Array Description File.txt”.
•	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): See table “cDNA Array Description File.txt”.
•	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. : Clones were obtained from Research Genetics (Huntsville, AL).
o	The method of reporter preparation: PCR products from these clones were prepared and printed onto glass slides according to previously described protocols (Khan et al., 1999b).
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.