astaxathin ( asx ) , a red natural pigment of the carotenoid family , has been suggested to play diverse biological roles in human health through its powerful antioxidant properties , .
recent studies have found that asx could also possess neuroprotective properties and might be useful against neurological disorders and in preventing the decline of cognitive functions , . nowadays ,
there has been a growing understanding that the influence of nutrient compound on health is complex and requires nutrigenomic studies to tease out the mechanism at the molecular level .
hence , we initiated to investigate asx - induced transcriptomic profiling on the adult hippocampus in a mouse model using a high - throughput dna microarray approach .
eleven - weeks - old male c57/bl6j mice were obtained from slc , co. , ltd .
the mice were housed individually in standard cages with a 12-h light / dark cycle .
after one - week of acclimatization rearing with standard powdered chow ( an mf diet , oriental yeast , tokyo , japan ) and water ad libitum , all mice were fed asx or its placebo powder ( astareal powder 20f based on 2% content of astaxanthin from haematococcus pluvialis ; fuji chemical industry , toyama , japan ) at final consistency of 0.1% , 0.5% and 0% ( control , hereafter abbreviated as con ) asx dose for four - weeks ( n  =  6/group ) .
all the experimental procedures were performed in accordance with protocols approved by the university of tsukuba animal experiment committee guidelines for the care and use of laboratory animals .
the sampling was performed a day after the last day of asx supplementation period to avoid acute influence . following the dissection of the brain , hippocampus ( right )
were carefully removed and immediately deep frozen in liquid nitrogen and stored at   80  c in a deep freezer .
the hippocampi were individually ground in liquid nitrogen to make very fine powders , and then their powders were stored in aliquots at   80  c till used for total rna extraction ( fig .
to obtain best results on the dna microarray chip , the critical assessment of high - quality rna is required .
total rna was extracted from the powdered samples using the qiagen rneasy mini kit ( qiagen , germantown , md , usa ) according to an optimized protocol , , . concentration and quality of total rna
were assessed a spectrometric method with nanophotometer ( implen , mnchen , germany ) and further confirmed using formaldehyde - agarose gel electrophoresis ( fig .
1b ) . in this experiment , we assessed a good quality of total rna in all samples as shown by a value of spectrophotometric absorbance ( via a260/280 and a260/230 ratio ) greater then 1.8 , respectively .
in addition , integrity of rna that comprised the 28s and 18s ribosomal rna is evaluated by formaldehyde - agarose gel electrophoresis stained with ethidium bromide as seen clearly on the gel images in fig .
an equal amount of total rna ( 1000  ng ) from four randomly chosen the powdered samples was pooled .
a mouse whole genome 4    44  k dna microarray chip ( agilent technologies , ca , usa ; g4122f ) was used for global gene expression into the hippocampus , where the two - color ( cy3 and cy5 labels ) in conjunction with dye - swap method , ,   was used ( fig .
the gene expression data from this study on asx supplementation effects in the adult mouse hippocampus were deposited in the gene expression omnibus ( geo ) database under the accession number gse62197 , and will be used for further analysis and study ( fig .
here we performed the quality control procedure based on the expression of two housekeeping genes ( gapdh and beta - actin ) by semi - quantitative reverse transcription - pcr ( rt - pcr ) for all samples ( n  =  4/group ) used in the dna microarray analysis .
for this , cdna was synthesized from independent samples using affinityscript qpcr cdna synthesis kit ( agilent technologies , la jolla , ca , usa ) according to the manufacturer 's protocol . the reaction mixture for rt - pcr
was prepared the emeraldamp pcr master mix ( takara shuzo , shiga , japan ) and then performed on thermal - cycling ( c1000 thermal cycler ; bio - rad laboratories , california , usa ) .
we validated that both the housekeeping genes were expressed at almost no differences among the groups ( p  >  0.05 ) ( fig .
2a ) ; the pcr amplified bands that were visualized and quantified using the image quant las 4000 mini ( ge healthcare , tokyo , japan ) are shown in fig . 2b .
furthermore , to achieve reliability of gene expression under dye - swap on the dna microarray approach , we confirmed the expression of beta - actin genes for all 10 probes on the level of signal intensity and fold change .
the control ( con ) and treated samples ( 0.1% and 0.5% asx ) were labeled with cy3 and cy5 , or with reversed cy5 and cy3 , respectively .
this result showed that the fluorescent signal differences did not alter due to our dye - swap approach , and the fold change on beta - actin probes appeared less than 1.5-fold ( fig .
2c ) , indicating that the expression of housekeeping gene for positive control is stable hybridization between reversed samples in the two - color dna microarray approach .
it is well known that an accurate outcome of differentially expressed genes from a microarray data requires the critical normalization of the hybridization intensity before the differentially expressed genes ( among treatments ) can be analyzed . in this study
, we used the filtering with replication based on the value of log2 ratio that can determine a systematic dependence on intensity spot in the two - color and dye - swap microarray analysis .
differentially up- and down - regulated genes using the cut - off values ( /  1.5/0.75-fold versus con ) were measured by con with cy5 dye and asx with cy3 dye in chip 1 , followed by a swap of the dye labeling in the chip 2 .
post - dye - swap and applied normalization , the number of overall genes is gradually reduced from the raw datasets ( 0.1% asx ; 44,480 genes , 0.5% asx ; 43,379 genes ) to a high confidence of differentially expressed gene list ( fig .
furthermore , the step of hybridization and washes with 60-mer probes was carried out based on the process in the agilent manufacturer 's guide , and the agilent scanner ( g2505c ) was used to detect the signals on the hybridized microarrays slide .
subsequently , the data for differential expression of genes was generated by agilent feature extraction software ( version 11.0.1.1 , agilent technologies ) .
as microarrays have been known to show several systematic errors including labeling bias , irregular feature morphologies , mismatched sample concentration and cross hybridization , in the agilent system these issues have been addressed by the development of the feature extraction algorithms .
to verify differentially expressed genes , as an example , we selected one gene , namely growth hormone ( gh ) gene that is highly up - regulated in both 0.1% ( 10.29-fold ) and 0.5% asx supplementations ( 4.29-fold ) and confirmed the validation for the expression between the employed two - color microarray approach ( fig .
4a ) and rt - pcr experiment using the pooled samples and individual samples ( fig . 4b and 4c )
. similar tendency of its expression levels was observed between the two experimental methods . to note , in the rt - pcr result using individual samples ( n  =  4/group )
, there was no significant difference among three groups from one - way anova ( p  >  0.05 ) , because it may be influenced by small number of samples and slight variation in each group ( fig .
nevertheless , a clear trend in rt - pcr is shown to be similar to the cy3 and cy5 values of the microarray method , which is mentioned a good relevance for positive control gene before .
this result demonstrates that a validation procedure using a discrete tool needs to recognize the quality of gene expression obtained in the microarray experiment .
thus , we sought to present and discuss the deposited microarray data set as a highly reliable dataset to understand the effect of asx supplementation on the hippocampus in our mouse model .
nutritional / dietary compounds may play an important role for brain structure and its function , and these effects would be mediated by changes in the genes .
dna microarray analysis is a powerful approach to identify genome - wide gene expression in response to intervention of nutrients on the central neuronal system referred to as nutrigenomics .
we performed this study with a long - term goal to investigate unique hippocampal gene profiling on the hippocampus of adult mouse affected by four - weeks of asx supplementation using whole - genome dna microarray analysis with the dye - swap methods ( agilent technologies ) . in this study , we show that the asx - induced genes have sufficient validity and reliability through a validation process including the quality control with housekeeping genes and the dye - swap based normalization .
therefore , our deposited dataset could be recognized as a first asx - influenced transcriptome analysis on the hippocampus of adult mouse .
furthermore , these large numbers of gene inventories will also provide a promising basis for understanding the complex molecular mechanisms that underlie neurobiological effect of asx on the hippocampus , the critical region for learning and memory , through highlighting potential candidate genes .
 naturally occurring astaxantin ( asx ) is one of the noticeable carotenoid and dietary supplement , which has strong antioxidant and anti - inflammatory properties , and neuroprotective effects in the brain through crossing the blood  brain barrier . 
 specially , we are interested in the role of asx as a brain food . although asx has been suggested to have potential benefit to the brain function , the underlying molecular mechanisms and events mediating such effect remain unknown . 
 here we examined molecular factors in the hippocampus of adult mouse fed asx diets ( 0.1% and 0.5% doses ) using dna microarray ( agilent 4    44  k whole mouse genome chip ) analysis . in this study , we described in detail our experimental workflow and protocol , and validated quality controls with the housekeeping gene expression ( gapdh and beta - actin ) on the dye - swap based approach to advocate our microarray data , which have been uploaded to gene expression omnibus ( accession number gse62197 ) as a gene resource for the scientific community . 
 this data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of asx supplementation on the brain , in particular the hippocampus . 
Direct link to deposited data
Experimental design, materials and methods
Workflow for optimal DNA microarray analysis through the quality assessment of total RNA
Confirmation of housekeeping gene expression between RT-PCR analysis and the obtained DNA microarray data
Hybridization, normalization, and scatter plot distribution of intensity values
Validation of DNA microarray data by re-confirmed RT-PCR result
Discussion