Table 1.

Interaction evidence types supported by myGRN

Evidence Type	Evidence Description	Score
Mined from Literature	Inference of an interaction from in silico textual analysis of a published paper by a natural language processing (NLP) algorithm.	0
Binding site/Promoter Interaction:
Transient transfection	Perturbation of expression of a reporter under control of the downstream gene promoter on disruption of upstream gene expression	10
Stable transgenic line	Perturbation of expression of a reporter under control of the downstream gene promoter on disruption of upstream gene expression	10
Protein synthesis inhibition	Demonstration of a direct interaction by inhibition of intermediate protein production using cyclohexamide or other inhibitors.	50
In silico prediction	Prediction of a binding site for the upstream gene product in the downstream gene promoter using in silico methods.	2
Elecrophoretic Mobility Shift Assay (EMSA)	EMSA showing direct binding between the upstream gene product and a fragment of the downstream gene promoter in vitro.	25
DNase Footprinting	Protection of a putative binding site in radiolabelled DNA containing the downstream gene promoter by the upstream gene product.	25
ChIP-on-chip	Chromatin Immunoprecipitation using antibody for the upstream gene, and analysis of the binding site locations on a microarray.	25
Expression Evidence:
Stable transgenic reporter	Transgenic organism carrying a reporter gene under the control of the promoter of the gene of interest.	25
Transient transgenic reporter	Transient transfection of a construct carrying a reporter gene under the control of the promoter of the gene of interest.	25
SAGE	Detection of a sequence tag from an mRNA in a Serial Analysis of Gene Expression experiment.	10
RT-PCR	Measurement of mRNA by Reverse-Transcription Polymerase Chain Reaction.	10
Primer extension	Extension of a gene-specific primer using an mRNA template.	10
Nuclease protection	Measurement of mRNA by hybridization with an antisense probe followed by ribonuclease digestion of unbound RNA.	10
Northern Blot	Measurement of mRNA by northern blot.	10
In situ hybridisation	Measurement of mRNA by in-situ hybridization.	25
cDNA Library	Isolation of a gene from a cDNA library.	10
Array	Measurement of mRNA on an expression DNA microarray.	10
Western blot	Measurement of protein by western blot.	10
Mass Spectrometry	Detection of protein from cell or tissue extract using mass spectrometry.	10
Immunohistochemistry/immunocytochemistry	Measurement of protein by antibody staining in tissue slices or cultured cells.	25
Perturbation Evidence:
Mutation of binding site	Site-directed mutagenesis of a known binding site of the upstream gene in the downstream gene's promoter leads to a decrease in activity of a reporter gene	25
In silico inference	Inference of an interaction from mathematical modelling of experimental data such as microarray time course.	2
Transient transgenic	Transient transfection of a DNA construct containing the upstream gene, controlled by a constitutive or inducible promoter.	10
Stable transgenic	Stable transgenic cell or organism line containing the upstream gene controlled by a constitutive or inducible promoter.	10
RNA-based overexpression	Introduction of synthetic or purified mRNA of upstream gene into target cells or tissues.	10
Protein-based overexpression	Introduction of synthetic or purified protein product of upstream gene to target cells or tissues.	10
Transgenic knock-out	Stable transgenic cell or organism line with the upstream gene knocked-out either functionally or by expression.	25
RNAi	RNA interference-based knock-down or silencing of the upstream gene.	25
Morpholino	Morpholino-based inhibition of translation of the upstream gene mRNA.	25
Molecular Inhibitor	Use of an inhibitor molecule to reduce or eliminate the function of the upstream gene product.	10
Dominant-negative protein expression	Insertion of a dominant-negative version of an upstream regulator into a system reduces or eliminates expression of the target gene.	10

There are 31 evidence types divided into three categories. Binding site evidence demonstrates a molecular interaction between the upstream gene product and the promoter of the downstream gene. Expression evidence shows that the interacting genes are co-expressed (activators) or inversely expressed (repressors) in a given tissue. Perturbation evidence shows that changes to the expression of the upstream gene lead to perturbed expression of the downstream gene. We have given each experimental evidence type one of four possible scores (50 – conclusive, 25 – strongly indicative, 10 – indicative or 2 – suggestive) reflecting how well it supports the appropriate category. Interaction evidence that is extracted from the literature with an NLP algorithm is not placed into any of these classes and does not contribute to the scoring of an interaction.