Were normalized using the Robust Multichip Average (RMA) algorithm using FlexArray software [27]. The normalized intensity values were referred to as units of intensity (UI). Genes expressed differently between the tumors and controls were identified using the algorithm Significance Analysis of Microarrays (SAM version 3.0, http://www.stat.stanford.edu/ tibs/SAM) using the cut-off values of a fold change (FC) of 1.5, a general false discovery rate (FDR) of 1 , and a local FDR of ,10 [28]. Unsupervised hierarchical clustering and principal component analysis (PCA) were performed using dChip software (version 1.6, www.dCHIP.org) and R language in Java’s platform, respectively.Validation of Global Gene Expression by a Second High throughput Microarray (HG-ST1.0)The gene expression profile of 24 samples explored with the HG-Focus microarray, including 19 CCs and 5 cervical epithelium controls, was also examined using the Human Gene 1.0 ST oligonucleotide microarray (Affymetrix, Santa Clara, CA). This array contains 33,297 probe sets that correspond to approximately 20,741 genes of the human gene reference database according to the UCSC Genome Browser Assembly Mar. 2006 NCBI 36/hg18, available at http://genome.ucsc.edu/. Total RNA preparationMitosis as Source of Biomarkers in Cervical Cancer(MW) non-parametric test. The correlations between the MA results and the qRT-PCR data were performed using log2 values and measured using Pearson’s correlation coefficient.ImmunohistochemistryThe protein expression of 10 genes was determined in 26 CC and 10 control samples with IH. Two homemade tissue microarrays (TMA) were built, one containing 14 HPV16positive CCs and 5 controls and the other 12 CC positive for other HPVs and 5 controls. NUSAP1 was explored only in samples of the first TMA. Cylindrical samples from representative regions of the paraffin embedded tissue blocks, previously selected by H E stained slides, were taken with a punch-biopsy needle (2 mm diameter), transferred to recipient paraffin blocks in defined array positions and newly embedded in paraffin. All the tissues blocks of matched patients were obtained from the Pathology Department of the hospital. Serial sections (4 mm thick) of the TMA were cut and the 10th slide was stained with H E to confirm the histopathological diagnosis. Sections were immersed in xylene to remove paraffin and then rehydrated with graded alcohol (100 , 95 , 90 , 18325633 80 , and 70 v/v in water). Epitope retrieval was performed by heating the slides, and introducing them into Target Retrieval Solution, pH 6.0 (Dako, Carpinteria, CA) at 121uC for 5 min in a pressure cooker. Endogenous peroxidase activity was blocked by incubating the slides with 1 hydrogen peroxide in PBS for 10 min. Then, a non-specific background blocker was added and incubated for 10 min. Primary Met-Enkephalin web antibodies against PCNA (sc-53407); p16 for CDKN2A (sc-71804); SCP-2 for SYCP2 (sc-20048), PRC1 (sc56345); cyclin B2 for CCNB2 (sc-81241); CDKN3 (sc-475); and CDC2 for p34 (sc-70822), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies against CDC20 (cat. 34?900), Ki-67 for MKI67 (cat. M7187) and NUSAP1 (cat. H00051203-B01) were obtained from Invitrogen, Dako (Glostrup, Denmark), and Nova Biological (Littleton, CO), respectively. The dilution used for all antibodies was 1:100, except for CDC2, (1:50) and NUSAP1 (1:250), and the antibody MedChemExpress 11089-65-9 diluent used was from Dako. A total volume of 300 mL was added to each section, and t.Were normalized using the Robust Multichip Average (RMA) algorithm using FlexArray software [27]. The normalized intensity values were referred to as units of intensity (UI). Genes expressed differently between the tumors and controls were identified using the algorithm Significance Analysis of Microarrays (SAM version 3.0, http://www.stat.stanford.edu/ tibs/SAM) using the cut-off values of a fold change (FC) of 1.5, a general false discovery rate (FDR) of 1 , and a local FDR of ,10 [28]. Unsupervised hierarchical clustering and principal component analysis (PCA) were performed using dChip software (version 1.6, www.dCHIP.org) and R language in Java’s platform, respectively.Validation of Global Gene Expression by a Second High throughput Microarray (HG-ST1.0)The gene expression profile of 24 samples explored with the HG-Focus microarray, including 19 CCs and 5 cervical epithelium controls, was also examined using the Human Gene 1.0 ST oligonucleotide microarray (Affymetrix, Santa Clara, CA). This array contains 33,297 probe sets that correspond to approximately 20,741 genes of the human gene reference database according to the UCSC Genome Browser Assembly Mar. 2006 NCBI 36/hg18, available at http://genome.ucsc.edu/. Total RNA preparationMitosis as Source of Biomarkers in Cervical Cancer(MW) non-parametric test. The correlations between the MA results and the qRT-PCR data were performed using log2 values and measured using Pearson’s correlation coefficient.ImmunohistochemistryThe protein expression of 10 genes was determined in 26 CC and 10 control samples with IH. Two homemade tissue microarrays (TMA) were built, one containing 14 HPV16positive CCs and 5 controls and the other 12 CC positive for other HPVs and 5 controls. NUSAP1 was explored only in samples of the first TMA. Cylindrical samples from representative regions of the paraffin embedded tissue blocks, previously selected by H E stained slides, were taken with a punch-biopsy needle (2 mm diameter), transferred to recipient paraffin blocks in defined array positions and newly embedded in paraffin. All the tissues blocks of matched patients were obtained from the Pathology Department of the hospital. Serial sections (4 mm thick) of the TMA were cut and the 10th slide was stained with H E to confirm the histopathological diagnosis. Sections were immersed in xylene to remove paraffin and then rehydrated with graded alcohol (100 , 95 , 90 , 18325633 80 , and 70 v/v in water). Epitope retrieval was performed by heating the slides, and introducing them into Target Retrieval Solution, pH 6.0 (Dako, Carpinteria, CA) at 121uC for 5 min in a pressure cooker. Endogenous peroxidase activity was blocked by incubating the slides with 1 hydrogen peroxide in PBS for 10 min. Then, a non-specific background blocker was added and incubated for 10 min. Primary antibodies against PCNA (sc-53407); p16 for CDKN2A (sc-71804); SCP-2 for SYCP2 (sc-20048), PRC1 (sc56345); cyclin B2 for CCNB2 (sc-81241); CDKN3 (sc-475); and CDC2 for p34 (sc-70822), were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies against CDC20 (cat. 34?900), Ki-67 for MKI67 (cat. M7187) and NUSAP1 (cat. H00051203-B01) were obtained from Invitrogen, Dako (Glostrup, Denmark), and Nova Biological (Littleton, CO), respectively. The dilution used for all antibodies was 1:100, except for CDC2, (1:50) and NUSAP1 (1:250), and the antibody diluent used was from Dako. A total volume of 300 mL was added to each section, and t.