Regassa et al. BMC Genomics 2011, 12:57 http://www.biomedcentral.com/1471-2164/12/57 reduce the variability in the concentration of the initial RNA populations across samples and to use equivalent RNA quantities to the ones used for array hybridization, 12 ng of the total RNA of each sample was used for cDNA synthesis using oligo (dt)25 and ran- dom primer as described elsewhere [58]. The ABI prism® 7000 apparatus (Applied Biosystems) was used to perform the quantitative analysis using SYBR® Green Jumpstart™ Tag Ready Mix™ (Sigma) incorporation for dsDNA specific fluorescent detection dye. Standard curves were fitted for both target genes and internal control (18S rRNA) using serial dilutions of plasmid DNAs containing 101-109 molecules and run in separate wells. PCR was assembled using 20 μl total reaction volume containing double distilled (dd) water, forward and reverse primers, SYBR green universal master mix (Sigma) and 2 μl template cDNAs using five replicates for each sample. During each reaction, samples from the same cDNA were run in duplicate to control the repro- ducibility of the results. A universal thermal cycling parameter (initial denaturizing step at 95°C for 3 min, 45 cycles of denaturizing at 95°C for 30 sec and 58°C for 30 sec were used to quantify mRNA expression level. After the end of the last cycle, dissociation curves were fitted by starting the fluorescence acquisition at 60°C and taking measurements every 7 sec interval until the temperature reaches 95°C. Final quantitative analysis was done using relative standard curve method and the expression values of the target transcripts were normal- ized to that of 18S. The mean normalized data were reported as the amount of a given target gene transcript in the two samples compared and significantly different means were identified using t-test (SPSS Inc. Chicago, IL, USA). Pairs of primers that were used for array vali- dation are shown in Table 1. The appropriateness of 18S as internal control was confirmed by the results of semi-quantitative RT-PCR showing its stability across all samples. Immunofluorescence staining In order to localize the proteins of some differentially expressed transcripts, ovarian sections were washed three times in PBS and fixed in 4% (w/v) Paraformalde- hyde overnight at 4°C. The fixed specimens were permeabilized during 2.5 hr incubation in 0.5% (v/v) Triton-X100 (Sigma) in PBS. To inhibit NSB of the anti- bodies, samples were subsequently blocked in 3% (w/v) bovine serum albumin (BSA) in PBS for 1 hr. The sec- tions were then incubated for 1 hr at 39°C and mounted onto glass slides with gelvatol. The primary antibody for IRF6 (rabbit anti-human polyclonal antibody, Santa Cruz Biotechnologies Inc., Germany) was used at 1:100 in PBS and for MSX1 (rabbit anti-human polyclonal Page 5 of 20 antibody, Lifespan Biosciences, USA) at 1:50 in blocking solution. The samples were incubated for 15 and 1 hr with primary and secondary antibodies (FITC conju- gated goat anti-rabbit secondary antibody (Lifespan Biosciences, USA), respectively using 1:100 ratio in both cases. Negative controls were processed in the same manner by omitting the use of primary antibody. In order to visualize the nucleus, the sections were finally incubated in 0.1 mg/ml 4’-6-Diamidino-2-phenylindole (DAPI, Sigma) or propidium iodide (Sigma). After the final wash in PBS, the sections were mounted on glass slides and visualized on ApoTome microscope (Apo- Tome MicroImaging, Inc., Carl-Zeiss, Germany). Results Specific transcription programs are exhibited by bovine oocytes and CCs In order to get an insight into specific transcription pro- gram in bovine oocytes and CCs, we analyzed transcrip- tome profile of GV (number 1) and MII (number 4) oocytes and their companion CCs (number 2 and 3, respectively) using MAS 5 present or absent call as described elsewhere [42,59]. The raw data from all arrays are available online at http://www.ncbi.nlm.nih.gov/geo/ with GEO accession number GSE21005. The analysis showed that of 13162 detected genes, 1516 and 2727 are exclusively expressed in GV oocytes (number 1) and their companion CCs (number 2), respectively while 8919 are expressed in both (Additional files, 2, 3 and 4). Similarly, of 13602 detected genes, 1423 and 3100 are expressed exclusively in MII oocytes (number 4) and their companion CCs (number 3), respectively and 9079 are expressed in both (Additional files, 5, 6 and 7). In addition, expression analysis of these detected genes showed that a total of 8612 transcripts are differentially expressed between GV oocytes (number 1) and CCs (number 2) of which 4304 and 4308 are over expressed in oocytes and CCs, respectively (Additional file 8). Simi- larly, a total of 8863 transcripts are differentially expressed between MII oocytes (number 4 ) and CCs (number 3) of which 4271 and 4592 were over expressed in MII oocytes and CCs, respectively (Additional file, 9). The heat map and hierarchical clustering of some of the top differentially expressed genes between oocytes and CCs at the two stages are presented in Figures 3A and 3B and 4A and 4B. The GO categories (biological and molecular functions) of transcripts over expressed in GV oocytes and CCs relative to each other are shown in Figure 5 and additional file 10, respectively. The most significantly changed molecular and cellular functions associated with genes over expressed in MII CCs relative to their enclosed oocytes and vice versa are also shown in additional file 11.