O select 
the most appropriate reference gene, seven different housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), b2-microglobulin (B2M), beta-actin (ACTB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), 18SmRNA expression microarray analysisTotal RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Quantity and quality of the isolated RNA were tested by measuring the absorbance and capillary gelelectrophoresis using the 2100 Bioanalyzer and RNABiomarkers for Dysplasia-Carcinoma TransitionTable 1. Number of patients per disease group participating in the study.GroupOriginal set Affymetrix microarrays (GSE4183, GSE10714)Independent set Affymetrix microarrays GSE37364 16 13 14 13 38 94 Array real-time PCR 13 11 10 10 4 20Adenoma with low-grade dysplasia High-grade dysplastic adenoma CRC Dukes A CRC Dukes C CRC with unknown stage Healthy Control Total patient numbers doi:10.1371/journal.pone.0048547.t9 11 
10 12 11ribosomal RNA (18S), tyrosine 3-monooxygenase/tryptophan 5monooxygenase activation protein, zeta polypeptide (YWHAZ)) were used on the real-time PCR array.X logit ln (P=?{P b0 zb1 DCt1 zb2 DCt2 z::::zbn DCtnStatistical evaluation of RT-PCR resultsRelative quantifications of the gene expression were performed and the fold change values were calculated using the DDCT method. The ASP015K chemical information threshold cycle (CT) of the 18S ribosomal RNA endogenous control was used to normalize target gene expression (DCT) to correct for experimental variation. Logistic regressions were applied to analyze dependence of binary diagnostic variables (represented 0 as control, 1 as disease) on the DCt values from the training set. When P (probability of a patient sample) is diagnosed 18055761 as “diseased,” then a function X = logit (P) can be defined as follows: Table 2. Real-time ready assays applied in RT-PCR validation.Maximum-likelihood fitting method was used to obtain the (empirical) coefficients bi that define the relationship between X and the experimental measurements DCti. The bi values were obtained using MedCalc software program (MedCalc Software). Receiving operating characteristic (ROC) curve analysis was applied to evaluate the discriminatory power of the gene panels [17]. Discriminant and principal component analysis were performed. Discriminant analysis was used primarily in order to predict membership of distinct groups. As a result “Classification results” tables were prepared showing a summary for subjectsAssay ID 103015 100950 103133 103136 103109 105522 103070 103045 103035 103210 103167 101128 102065 102488 102079 102119 104092Gene Symbol CA7 IL1B IL1RN IL8 GREM1 CXCL1 CXCL2 COL12A1 CHI3L1 SLC7A5 MMP3 GAPDH B2M ACTB HPRT1 RPL13A RN18S1 YWHAZGene name carbonic anhydrase VII interleukin 1, beta interleukin 1 Fexinidazole receptor antagonist interleukin 8 gremlin 1 chemokine (C-X-C motif) ligand 1 chemokine (C-X-C motif) ligand 2 collagen, type XII, alpha 1 chitinase 3-like 1 solute carrier family 7, member 5 matrix metallopeptidase 3 glyceraldehyde-3-phosphate dehydrogenase beta-2-microglobulin actin, beta hypoxanthine phosphoribosyltransferase 1 ribosomal protein L13a RNA, 18S ribosomal 1, 18S ribosomal RNA Top of Form tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptideAmplicon length 77 87 76 92 111 105 95 66 76 72 110 112 76 102 102 124 73Position 416?92 162?48 343?18 879?70 144?54 340?44 431?25 2287?352 433?07 1500?.O select the most appropriate reference gene, seven different housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), b2-microglobulin (B2M), beta-actin (ACTB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), 18SmRNA expression microarray analysisTotal RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Quantity and quality of the isolated RNA were tested by measuring the absorbance and capillary gelelectrophoresis using the 2100 Bioanalyzer and RNABiomarkers for Dysplasia-Carcinoma TransitionTable 1. Number of patients per disease group participating in the study.GroupOriginal set Affymetrix microarrays (GSE4183, GSE10714)Independent set Affymetrix microarrays GSE37364 16 13 14 13 38 94 Array real-time PCR 13 11 10 10 4 20Adenoma with low-grade dysplasia High-grade dysplastic adenoma CRC Dukes A CRC Dukes C CRC with unknown stage Healthy Control Total patient numbers doi:10.1371/journal.pone.0048547.t9 11 10 12 11ribosomal RNA (18S), tyrosine 3-monooxygenase/tryptophan 5monooxygenase activation protein, zeta polypeptide (YWHAZ)) were used on the real-time PCR array.X logit ln (P=?{P b0 zb1 DCt1 zb2 DCt2 z::::zbn DCtnStatistical evaluation of RT-PCR resultsRelative quantifications of the gene expression were performed and the fold change values were calculated using the DDCT method. The threshold cycle (CT) of the 18S ribosomal RNA endogenous control was used to normalize target gene expression (DCT) to correct for experimental variation. Logistic regressions were applied to analyze dependence of binary diagnostic variables (represented 0 as control, 1 as disease) on the DCt values from the training set. When P (probability of a patient sample) is diagnosed 18055761 as “diseased,” then a function X = logit (P) can be defined as follows: Table 2. Real-time ready assays applied in RT-PCR validation.Maximum-likelihood fitting method was used to obtain the (empirical) coefficients bi that define the relationship between X and the experimental measurements DCti. The bi values were obtained using MedCalc software program (MedCalc Software). Receiving operating characteristic (ROC) curve analysis was applied to evaluate the discriminatory power of the gene panels [17]. Discriminant and principal component analysis were performed. Discriminant analysis was used primarily in order to predict membership of distinct groups. As a result “Classification results” tables were prepared showing a summary for subjectsAssay ID 103015 100950 103133 103136 103109 105522 103070 103045 103035 103210 103167 101128 102065 102488 102079 102119 104092Gene Symbol CA7 IL1B IL1RN IL8 GREM1 CXCL1 CXCL2 COL12A1 CHI3L1 SLC7A5 MMP3 GAPDH B2M ACTB HPRT1 RPL13A RN18S1 YWHAZGene name carbonic anhydrase VII interleukin 1, beta interleukin 1 receptor antagonist interleukin 8 gremlin 1 chemokine (C-X-C motif) ligand 1 chemokine (C-X-C motif) ligand 2 collagen, type XII, alpha 1 chitinase 3-like 1 solute carrier family 7, member 5 matrix metallopeptidase 3 glyceraldehyde-3-phosphate dehydrogenase beta-2-microglobulin actin, beta hypoxanthine phosphoribosyltransferase 1 ribosomal protein L13a RNA, 18S ribosomal 1, 18S ribosomal RNA Top of Form tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptideAmplicon length 77 87 76 92 111 105 95 66 76 72 110 112 76 102 102 124 73Position 416?92 162?48 343?18 879?70 144?54 340?44 431?25 2287?352 433?07 1500?.
