Ntiate CC and CIN2/3 (CIN2+) from CIN1 and controls. Immunohistochemistry (IH) was performed for 10 selected proteins in 26 CC samples and 10 control samples. The association of 9 markers with survival was investigated by survival analysis of 42 patients with HPV16positive CC who were followed up for at least 42 months.DNA and RNA Isolation Subjects, Samples, and Experimental DesignThe study subjects included 69 patients with invasive cervical cancer (CC) diagnosed in the Department of Oncology, 29 patients with low-grade CIN (CIN1), 21 patients with high-grade CIN (CIN2 and CIN3), and 25 women with normal cervicalDNA was purified from cervical scrapes and some biopsy specimens using the PureLink Genomic DNA Kit (Invitrogen, Grand Island NY) and maintained at 220uC until analysis. Total RNA was isolated from one half of the divided biopsy using TRIzol reagent (Invitrogen), according to the manufacturer’sMitosis as Source of Biomarkers in Cervical Cancerprotocol. The Title Loaded From File quality of the RNA was confirmed by agarose gel electrophoresis, as demonstrated by the presence of Title Loaded From File intact ribosomal RNA, with the 28s band twice as intense as the 18s band.Detection and HPV TypingHPV detection was performed by PCR using universal primers located in the HPV L1 gene MY09/MY11, GP5+/6+, and L1C1 as described previously [23?5]. The HBB gene was used as an internal control to assess the quality of DNA. The HPV types were identified by sequencing the amplified bands in positive samples using a fluorescent cycle-sequencing method (BigDye Terminator Ready Reaction Kit, Applied Biosystems, Foster city, CA). Sequence analysis was performed using an ABI PRISM 3130xl genetic analyzer (Applied Biosystems). Each sequence from the HPV positive samples was analyzed with the FASTA sequence similarity tool [26]. The average percentage identity of these sequences to HPV types was 98.7 (range, 91?00 ).(300 ng), labeled DNA synthesis, hybridization, scanning, and image analysis were performed according to the manufacturer’s protocols (Affymetrix GeneChip Expression Assay manual). To assess the quality of the experiments, the following parameters were used: expression of the exogenous poly-A controls, the presence of oligo B2 used to make grid alignments, and area under the curve (AUC) values above 0.8. Only those microarrays with optimal quality controls were analyzed. Microarrays were normalized using the RMA algorithm in the Affymetrix 18325633 expression console. The normalized intensity values were referred to as units of intensity (UI). The normalized intensities (log2 values) of the 8,370 genes that were examined on both microarrays (HG ST1 and HG Focus) were compared, and the level of correlation was assessed with Pearson’s correlation coefficient.Validation of Global Gene Expression by Real-time Quantitative Retrotranscription PCR (qRT-PCR)Reverse transcription of total RNA was performed using the High-Capacity cDNA Archive kit (Applied Biosystems) in a total volume of 20 mL. The mix included 2 mg of RNA, 2 mL of 106 RT buffer, 0.8 mL of 100 mM dNTPs, 2 mL of 106 RT Random Primers, 1 mL of MultiScribeTM reverse transcriptase (5 U/mL), and 1 mL of RNase inhibitor (2 U/mL). Reactions were incubated at 37uC for 120 min, and then stored at 220uC. A set of 23 genes was used to validate gene expression in 44 HPV16-positive CC and 25 healthy cervical epithelium control samples with qRTPCRs using TaqMan probes. The genes included are CCNB2, CDC2, CDC20, CDKN2A, CDKN3, CKS2, MCM2, MKI67.Ntiate CC and CIN2/3 (CIN2+) from CIN1 and controls. Immunohistochemistry (IH) was performed for 10 selected proteins in 26 CC samples and 10 control samples. The association of 9 markers with survival was investigated by survival analysis of 42 patients with HPV16positive CC who were followed up for at least 42 months.DNA and RNA Isolation Subjects, Samples, and Experimental DesignThe study subjects included 69 patients with invasive cervical cancer (CC) diagnosed in the Department of Oncology, 29 patients with low-grade CIN (CIN1), 21 patients with high-grade CIN (CIN2 and CIN3), and 25 women with normal cervicalDNA was purified from cervical scrapes and some biopsy specimens using the PureLink Genomic DNA Kit (Invitrogen, Grand Island NY) and maintained at 220uC until analysis. Total RNA was isolated from one half of the divided biopsy using TRIzol reagent (Invitrogen), according to the manufacturer’sMitosis as Source of Biomarkers in Cervical Cancerprotocol. The quality of the RNA was confirmed by agarose gel electrophoresis, as demonstrated by the presence of intact ribosomal RNA, with the 28s band twice as intense as the 18s band.Detection and HPV TypingHPV detection was performed by PCR using universal primers located in the HPV L1 gene MY09/MY11, GP5+/6+, and L1C1 as described previously [23?5]. The HBB gene was used as an internal control to assess the quality of DNA. The HPV types were identified by sequencing the amplified bands in positive samples using a fluorescent cycle-sequencing method (BigDye Terminator Ready Reaction Kit, Applied Biosystems, Foster city, CA). Sequence analysis was performed using an ABI PRISM 3130xl genetic analyzer (Applied Biosystems). Each sequence from the HPV positive samples was analyzed with the FASTA sequence similarity tool [26]. The average percentage identity of these sequences to HPV types was 98.7 (range, 91?00 ).(300 ng), labeled DNA synthesis, hybridization, scanning, and image analysis were performed according to the manufacturer’s protocols (Affymetrix GeneChip Expression Assay manual). To assess the quality of the experiments, the following parameters were used: expression of the exogenous poly-A controls, the presence of oligo B2 used to make grid alignments, and area under the curve (AUC) values above 0.8. Only those microarrays with optimal quality controls were analyzed. Microarrays were normalized using the RMA algorithm in the Affymetrix 18325633 expression console. The normalized intensity values were referred to as units of intensity (UI). The normalized intensities (log2 values) of the 8,370 genes that were examined on both microarrays (HG ST1 and HG Focus) were compared, and the level of correlation was assessed with Pearson’s correlation coefficient.Validation of Global Gene Expression by Real-time Quantitative Retrotranscription PCR (qRT-PCR)Reverse transcription of total RNA was performed using the High-Capacity cDNA Archive kit (Applied Biosystems) in a total volume of 20 mL. The mix included 2 mg of RNA, 2 mL of 106 RT buffer, 0.8 mL of 100 mM dNTPs, 2 mL of 106 RT Random Primers, 1 mL of MultiScribeTM reverse transcriptase (5 U/mL), and 1 mL of RNase inhibitor (2 U/mL). Reactions were incubated at 37uC for 120 min, and then stored at 220uC. A set of 23 genes was used to validate gene expression in 44 HPV16-positive CC and 25 healthy cervical epithelium control samples with qRTPCRs using TaqMan probes. The genes included are CCNB2, CDC2, CDC20, CDKN2A, CDKN3, CKS2, MCM2, MKI67.
