Total RNA was extracted from each of the frozen organs of eight animals per experimental group with TRIzol reagent (Invitrogen, Breda, The Netherlands), followed by a repeated phenol-chloroform extraction, LiCl precipitation, and additional purification using the RNeasy Mini Kit (Qiagen Benelux, Venlo, The Netherlands). The RNA quality was assessed using the RNA 6000 Nano LabChip® Kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA). All samples had intact bands corresponding to 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had an RNA integrity number (RIN) > 8. Five out of eight animals per group were chosen based on the best RNA quality across all 5 tissues, so that the RNAs used in this study all originated from the same 5 mice per time point.
Label
Cy3
Label protocol
1,5 µg of total RNA was amplified using the Amino Allyl MessageAmp aRNA kit (Ambion, Austin, Texas, USA), and labeled with Cy5 (experimental samples) and Cy3 (common reference) reactive dye according to the manufacturer’s instructions.
Total RNA was extracted from each of the frozen organs of eight animals per experimental group with TRIzol reagent (Invitrogen, Breda, The Netherlands), followed by a repeated phenol-chloroform extraction, LiCl precipitation, and additional purification using the RNeasy Mini Kit (Qiagen Benelux, Venlo, The Netherlands). The RNA quality was assessed using the RNA 6000 Nano LabChip® Kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA). All samples had intact bands corresponding to 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had an RNA integrity number (RIN) > 8. Five out of eight animals per group were chosen based on the best RNA quality across all 5 tissues, so that the RNAs used in this study all originated from the same 5 mice per time point.
Label
Cy5
Label protocol
1,5 µg of total RNA was amplified using the Amino Allyl MessageAmp aRNA kit (Ambion, Austin, Texas, USA), and labeled with Cy5 (experimental samples) and Cy3 (common reference) reactive dye according to the manufacturer’s instructions.
Hybridization protocol
The microarrays were hybridized overnight with 200 µl hybridization mixture, consisting of 50 µl Cy3-and Cy5-labeled aRNA , 100 µl Formamide and 50 µl 4 x RPK0325 MicroArray Hybridization Buffer (Amersham Pharmacia Biosciences, Piscataway, NJ, USA) at 37°C and washed in an Automated Slide Processor (Amersham Pharmacia Biosciences, Piscataway, NJ, USA)
Scan protocol
Scanned on an Agilent G2565AA scanner. Spot intensities were quantified as artifact removed densities, using Array Vision software (version 6.0)
Description
Common-reference sample was a pool of equal amounts of RNA from all the samples investigated, including additional samples from 5 mice that were fasted for 48 hours supplemented with vitamin B complex after 24 and 36 hours of fasting.
Data processing
Estimated foreground and background signals were extracted from the ArrayVision files. Quality control was performed on the raw data; two arrays did not pass our quality criteria because of spatial artifacts and one array (from liver and clustering with brain) was excluded from further analysis. Background correction was performed by using the normexp method (Bioconductor package limma) to adjust the foreground signal without introducing negative values. Resulting log-ratios were normalized per array by using print-tip loess. Control probes were downweighted to zero in the normalization step. The oligo library was re-annotated by aligning each oligo to the Ensembl assembly using a BLAT/BLAST search. If no hit was found, oligos were blasted to a set of sequences containing Refseq RNAs (version 22) and UniGene clusters (version 163) that could not be aligned with the genome. Using information from NCBI Refseq and Unigene identifierss were linked to an Entrez gene ID. These Entrez gene IDs were used as input for the Bioconductor package AnnBuilder, with which a annotation data package was created.
