|Application:||Gene expression microarray analysis|
|Number of samples:||30|
|Release date:||Jul 7 2008|
|Last update date:||May 10 2018|
|Diseases:||Deficiency Diseases, Anemia, Iron-Deficiency|
|Dataset link||Liver expression of Bmp6, Smad7, Id1 and Atoh8 is regulated by iron stores|
Total RNA was extracted and purified using the RNeasy Lipid Tissue kit (Qiagen, Courtaboeuf, France). RNA quality was checked on RNA 6000 Nano chips using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). RNA samples used for chip experiments all had RNA Integrity Numbers (RIN) 16 greater than 9. Agilent’s Low RNA Input Linear Amplification Kit PLUS (One-color) was used to generate fluorescent cRNA. The amplified cyanine 3-labeled cRNA samples were then purified using Qiagen’s RNeasy mini spin colums and hybridized to Agilent Whole Mouse Genome Microarrays, 4x44K. Microarray slides were washed and scanned with an Agilent Scanner, according to the standard protocol of the manufacturer. Information from probe features was extracted from microarray scan images using the Agilent Feature Extraction software v.9.5.1. All the analyses were performed using Bioconductor, an open source software for the analysis of genomic data rooted in the statistical computing environment R. Normalization between arrays was performed using the quantile method. Genes for which the background-corrected signal intensities were not greater than 2.6 standard deviations above the average background in at least 3 B6 and 3 D2 mice were assumed not to be expressed in the liver and were excluded from further analysis. The R/MAANOVA package implemented in Bioconductor was used to perform a two-way analysis of variance in which the log2-transformed expression level was considered to be a function of diet, strain, and the effects of the interaction between these two factors. Fs tests, based on the James-Stein shrinkage estimates of the error variance, were computed on a gene-by-gene basis, and p values obtained by permutation analysis. The proportion of false positives among all the genes initially identified as being differentially expressed (FDR) was assessed using the procedure described by Storey. When influence of diet on expression levels was significant, t-tests were performed to investigate specific effects of iron deficiency and iron enrichment.