truncatula 2HA and line 2HA and its progenitor Jemalong at day 0 and day 14. They were grown on medium selleck chemical containing 10M NAA and 4M BAP. At two weeks, callus cells start to proliferate and there are no morphological differences can be seen between the 2HA and Jemalong. Thus, two weeks pro vides an early time point to compare proliferating cultures of these two lines. The first Inhibitors,Modulators,Libraries appearance of embryos in 2HA occurs after five weeks of culture, but not in Jemalong. To investigate gene expression profiles and their changes during the early stage of regeneration, we profiled and compared the transcriptomes of 2HA and Jemalong, by extracting total RNA from three independently grown two week old cultures and analysing them on Affymetrix Medicago genome arrays. An average of 46%.

There were signifi cant similarities Inhibitors,Modulators,Libraries between 2 fold cut off method and SAM two class unpaired analysis. One hundred ninety five probe sets were up regulated in the embryogenic culture by both analyses while 38 probe sets were down regulated. The full data set has been deposited in the Gene Expression Omnibus database as accession GSE8131 and the normalised data set is available in the additional file 3. Array verification Quantitative real time RT PCR was used to confirm the level of expression of 10 transcripts from the array. For all probe sets tested, the expression ratios dis played the same pattern of expression as the array normal ised data but with amplified fold changes. For instance, probe set Mtr. 10439. 1. S1 at showed down reg ulation by real time RT PCR in the embryogenic culture, consistent with the array data.

In average, the fold changes in RT PCR data were approximately three times higher than that of array data indicating amplification of fold changes by sensitive real time RT PCR analysis. Inhibitors,Modulators,Libraries However, the fold changes were much closer to array un normalised data indicating Inhibitors,Modulators,Libraries normalisation may considera bly reduce the signal differences. The functional signifi cance of the transcripts validated by qRT PCR is discussed in more details below. Functional classification of differentially expressed probe sets The Medicago genome Inhibitors,Modulators,Libraries array does not incorporate the entire M. truncatula genome, it was created based on an incomplete genome sequence and ESTs from the Medicago truncatula Gene Index.

We have noted the inclu sion of probe sets for IMGAG gene predictions and the sellectchem corresponding EST leading to a duplication of data, and the absence of some consensus ESTs from MtGI available at the time the chip was made and also incorrect annota tion of some genes in both IMGAG and MtGI. Annotation of the probe sets on the Genome array also varies widely in quality. To interpret the gene expression data better, we have used GeneBins to provide hierarchical functional classification modelled on KEGG ontology. This analysis made it apparent that the metabolism seems to be different between the embryo genic and the non embryogenic M. truncatula cultures.