Supplementary Table S1. Microarray data analysis methods: Different analytical and statistical methods are usedto evaluate the immense datasets derived from high-throughput biological studies. Methods used for microarray data analysis are shown as representative.
Type of analysis / Statistical methodCo-expression analysis / Hierarchical clustering
Partitional clustering (e.g. k-means clustering)
Dimensionality reduction and class separation / Principal component analysis
Linear discriminant analysis
Pattern recognition in datasets / Supervised/unsupervised machine learning
Self-organizing maps
Reconstruction of transcriptional networks / Boolean network modeling
Modeling based on differential equations
Bayesian networks analysis
Supplementary Table S2. Brief description of technologies used in systems biological research.
cDNA/oligonucleotide microarrays. Spotted oligonucleotides or cDNAs are arrayed on a solid background. The sequence of nucleotides in each spot is complementary to a known mRNA/gene sequence. Hybridization of labeled cRNA derived from cells/tissues generates signals whose intensities reflect the concentration of specific mRNA species.
Serial analysis of gene expression (SAGE). A method for high-throughput analysis of mRNA abundance in cells/tissues. RNA is extracted from the cells/tissues and small tags are generated from each mRNA molecule. Concatamers of these small tags are generated with the number of tags incorporated reflecting the abundance of the original mRNA species. Cloning and sequencing of the concatamers will determine the number of incorporated tags of each sequence, which will represent the abundance of original mRNA.
Two-dimensional gel electrophoresis. A well-established method for separating complex mixtures of proteins based on two physicochemical properties; isoelectric point and molecular mass. Electrophoresis across a gradient of pH leads to separation of proteins based on their isoelectric points. In the second dimension which is basically a regular SDS-PAGE, proteins with the same isoelectric point will be further separated based on their molecular weights.
Mass spectrometry: Analyzing the composition of macromolecules (e.g. proteins) of a mixture by measuring the mass to charge ratio (m/z) of ions generated from constituents (e.g. peptides). Time of flight (TOF) is a common method of mass spectrometric analysis which uses the time an accelerated ion needs to reach the detector to calculate the m/z ratio.
Matrix-assisted laser desorption ionization (MALDI). An ionization technique used prior to mass spectrometric analyses of biomolecules. The sample is absorbed to a matrix and then ionized/vaporized using a laser beam. Matrix is designed to efficiently absorb the laser beam, while protecting the analyte from destruction and facilitating its ionization/vaporization. MALDI is usually used in combination with TOF mass analyzers.
Gas chromatography-mass spectrometry (GC-MS). Mass spectrometry combined with gas chromatography as a separation technique.
Liquid chromatography-mass spectrometry (GC-MS). Mass spectrometry combined with liquid chromatography as a separation technique.
Stable isotope labeling with amino acids in cell culture (SILAC). A mass-spectrometry-based method to compare protein quantities in two different cell culture samples. The cells whose proteomes are to be compared are cultured in parallel, with one of them being exposed to amino acids containing a stable heavy isotope. Proteins derived from the two sources are combined and simultaneously analyzed by mass spectrometry. Due to isotope incorporation in one of the samples, corresponding peptides can be differentiated by a shift in M/Z ratio with the relative peak intensities representing the relative protein abundance in samples.
Yeast two-hybrid: A method for detection of protein-protein interactions at a proteome scale. The assay relies on the fact that the two domains of a transcription factor, i.e. DNA-binding domain and activation domain, need to be in close proximity to drive gene expression from a reporter gene. Cloning two sets of proteins upstream of each domain and cotransfection of yeast cells with both vectors will result in reporter gene expression only in a fraction of cells in which the two domains are in physical proximity, i.e. via interacting proteins. Isolation of positive clones and sequencing of vectors will identify the pair of interacting proteins.
Chip-CHIP analysis: a method for high-throughput detection of protein-DNA interactions. Cells are transfected with a vector expressing a tagged transcription factor which will bind the target DNA sequence in the cells. Cells are treated with a crosslinking reagent, nuclei are isolated and fragments of DNA will be subjected to immunoprecipitation with tag-specific antibody. Precipitated DNA is purified, amplified/labeled and hybridized to a DNA microarray to detect sites of transcription factor binding.
DamID: a method for high-throughput detection of protein-DNA interactions. A transcription factor is expressed as a fusion protein with DNA-methyl transferase (Dam). This leads to methylation of specific DNA binding sites which is followed by digestion of DNA with methylation-specific restriction enzyme DpnI and purification, amplification, labeling and hybridization of methylated DNA fragments to a DNA microarray.
Antibody/protein arrays: Antibodies targeting different antigens are arrayed on a solid background. Antibodies at each spot capture different antigens from a protein lysate which can be subsequently quantified. Protein arrays are used similarly to detect protein-protein interactions.