Comment[GEAAccession]	E-GEAD-326
MAGE-TAB Version	1.1
Investigation Title	Systematic Expression Profiling of the Mouse Transcriptome Using RIKEN cDNA Microarrays
Experiment Description	The number of known mRNA transcripts in the mouse has been greatly expanded by the RIKEN Mouse Gene Encyclopedia project. Validation of their reproducible expression in a tissue is an important contribution to the study of functional genomics.     To assign absolute expression values for mouse transcripts, we applied the E17.5 reference sample to CAGE (cap analysis of gene expression) and expressed sequence tag (EST) high-throughput tag sequencing.     We add a reliability index named SRED (Spot Reliability Evaluation Score for DNA microarrays) for each spot. The SRED scores represent the probability that the calibrated gene expression level from a DNA microarray would be less than a factor of 2 different from that of quantitative real-time polymerase chain reaction assays whose dynamic quantification range is treated statistically to be similar to that of the DNA microarray.
Experimental Design	development or differentiation design	organism part comparison design
Experimental Factor Name	tissue
Experimental Factor Type	tissue
Person Last Name	Hayashizaki
Person First Name	Yoshihide
Person Affiliation	Laboratory for Genome Exploration Research Group,Genomic Sciences Center,The Institute of Physical and Chemical Research (RIKEN)
Person Roles	submitter
Public Release Date	2019-09-24
PubMed ID	11226216	12819129	14960301	15788151
Protocol Name	P-GEAD-266	P-GEAD-267	P-GEAD-268	P-GEAD-269	P-GEAD-270	P-GEAD-271
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol
Protocol Description	Frozen stock in water at -80 degree C	AGPC method	Deoxynucleotides labeled with the dyes Cy3 and Cy5 were obtained from Amersham Pharmacia. The labeling was carried out at 42 degree C for 1 h in a total volume of 30 microl containing 400 units of SuperScript II 0.1 mM Cy3-dUTP (or Cy5-dUTP); 0.5 mM each dATP, dCTP, and dGTP; 0.2 mM dTTP, 10 mM DTT, 6 microl of 5x first-strand buffer, 1 microg of mRNAs and 6 microg of random primers. The double step labeling method with amino-allyl dUTP. After incubation of 25 microg of total RNAs with 4 microg of oligo dT primer at 70  degree C for 10 min, 6 microl of 5x first strand buffer, 3 microl of 0.1 M DTT, 1.5 microl of the mixture of dGTP, dATP, and dCTP (10 mM each), 3.6 microl of 2.5 mM dTTP, 2.4 microl of 2.5 mM amino-allyl dUTP and 2 microl of SuperScript II reverse transcriptase was added and incubated at 42  degree C for 1 h.  Reverse transcription was stopped by the addition of 1.5 microl of 20 mM EDTA and then 1.5 microl of 1 N NaOH were added and incubated at 70  degree C for 10 min to hydrolyze RNA. The samples were neutralized by adding 1.5 microl of 1 N HCl and purified using a GFX PCR DNA and Band Purification Kit. After purification, DNA preparations were dried using a Speedvac concentrator and dissolved in 9 microl of 0.1 M sodium bicarbonate buffer (pH 9.0) for dye labeling. Cy3 and Cy5 mono-functional reactive dyes were prepared with diluting by 45 microl of DMSO and 2.8 microl was used for each reaction. Coupling was performed with the mixture of the dried cye dye and sodium bicarbonate-diluted sample at room temperature in darkness for 1 h.  Then reactions were quenched by addition of 4.5 microl of 4 M hydroxylamine at room temperature in the dark for 15 min.	Hybridized in a final volume of 30microl. Prior to hybridization, probe aliquots were heated at 95 degree C for 1 min and cooled at room temperature. Cover slips were hybridized overnight at 65 degree C in a hybricasette (obtained from ArrayIt.com). After hybridization, slides were washed in 2 x SSC, 0.1% SDS until the cover slips dropped off, the slides were then transferred into 1 x SSC, shaken gently for 2 min, and rinsed with 0.1 x SSC for 2 min. After washing, slides were spun at 800 rpm using a SORVALL (RC-3B plus; rotor, H6000A/HBB6) centrifuge.	scanned on a ScanArray 5000 confocal laser scanner, analyzed by using IMAGENE (BioDiscovery; Los Angeles), analyzed by using Digital GENOME (MolecularWare; California). The DNA solution was spotted on polylysine-coated slides (Corning Inc.) by using a DNA arrayer (http://cmgm.stanford.edu/pbrown/mguide/index.html). The diameter of the spots was 100-150 microm. Mouse beta-actin and G3PDH cDNAs were used as positive controls, and Arabidopsis cDNAs were used as negative controls (Accession nos. X98108 [GenBank] , X13611 [GenBank] ,X90769 [GenBank] , Z99707 [GenBank] , AF004393 [GenBank] , Z49777 [GenBank] , Q03943 [GenBank] , U58284 [GenBank] ). The DNA solution was spotted on aminosilane-coated slides (Corning Inc.) by using a DNA arrayer (http://cmgm.stanford.edu/pbrown/mguide/index.html) with 48 tips (SMP3, TeleChem International). The diameter of the spots was 100-150 microm. Mouse beta-actin and G3PDH cDNAs were used as positive controls, and Arabidopsis cDNAs were used as negative controls (Accession nos. X98108 [GenBank] , X13611 [GenBank] , X90769 [GenBank] , Z99707 [GenBank] , AF004393 [GenBank] , Z49777 [GenBank] , Q03943 [GenBank] , U58284 [GenBank] ).	normalized to the reference standard by subtracting (in log space) the median observed value if it were other than zero A filtering program, PRIM (Preprocessing Implementation for Microarray); Briefly, this program (i) deletes the results with \"flags\" added manually to corrupted spots, (ii) eliminates spots with signal intensities less than the mean + 3 x standard deviation of the background signal intensity in either Cy3 or Cy5, and (iii) eliminates spots located outside the least-mean-squares line +-2 x standard deviation. After the filtering was finished, we compared the results of the two experiments by calculating a Pearson's correlation coefficient. If the coefficient was equal to or greater than 0.7, we used the data in subsequent analyses. Improved PRIM; This procedure consisted of three steps; 1) eliminate spots that were flagged manually; 2) eliminate spots whose signal intensity is less than microbg + x bg (x 0) in both channels, where microbg and bg are the mean and standard deviation of the background signal intensity; 3) eliminate spots more than y from the least-mean-square line. The parameters x and y were determined so that the value of NR has the maximum score (Smax) at each step. After the filtering was finished, we compared the results of the two experiments by calculating a Pearson's correlation coefficient. If the coefficient was equal to or greater than 0.7, we used the data in subsequent analyses.
SDRF File	E-GEAD-326.sdrf.txt
Comment[Number of channel]	dual-channel
Comment[Array Design REF]	A-GEAD-5
Comment[AEExperimentType]	transcription profiling by array
Comment[SecondaryAccession]	CBX5
Comment[BioProject]	PRJDB8787
Comment[CIBEX Accept Date]	2004-08-17
Comment[Last Update Date]	2019-09-26