The nearest shrunken centroid method (Prediction Analysis for miroarrays �C PAM) was applied for sample classification from gene expression data. The pre-processing, data mining and statistical steps were performed using R-environment with Bioconductor libraries. selleck Vismodegib Hierarchical cluster analysis represents on each comparisons of correlation. Logistic regression was applied to analyze dependence of binary diagnostic variables (represented 0 as control, 1 as disease). Discriminant and principal component analysis were also performed. In the discriminant analysis, leave-one out classification was applied for cross-validation. Array real-time PCR Commercially available real-time PCR assays were applied for expression measuring of 11 discriminatory transcripts (www.roche-applied-science.com).

The list of the real-time ready assays can be seen in the Table 2. Gene specific forward and reverse primers and fluorescently labeled hydrolysis probes from Universal ProbeLibrary (F. Hoffmann-La Roche Ltd., Switzerland, Basel) were lyophilized into wells of 384-well PCR plates. Using Transcriptor First Strand cDNA Synthesis Kit (Roche), 2.5 ��g total RNA from 20 healthy, 24 adenoma, 24 CRC biopsy samples were reverse transcribed (Table 1). The quality of the cDNA samples was checked by real-time PCR for SDHA (succinate dehydrogenase complex, subunit A, flavoprotein) housekeeping gene. The expression analysis of the selected genes was performed from 5 ng/sample cDNA template, using the newly designed array real-time PCR cards and LightCycler 480 Probes Master (Roche).

The measurements were performed using a LightCycler 480 instrument as described in the products User Guide (http://www.roche-applied-science.com). After enzyme activation and denaturation at 95��C for 10 min, 45 PCR cycles were performed (denaturation at 95��C for 10 sec, annealing and extension at 60��C for 30 sec and signal detection at 72��C for 1 sec). In order to select the most appropriate reference gene, seven different housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ��2-microglobulin (B2M), beta-actin (ACTB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), 18S ribosomal RNA (18S), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)) were used on the real-time PCR array.

Table 2 Real-time ready assays applied in RT-PCR validation. Statistical evaluation of RT-PCR results Relative quantifications of the gene expression were performed and the fold change values were Carfilzomib calculated using the ����CT method. The threshold cycle (CT) of the 18S ribosomal RNA endogenous control was used to normalize target gene expression (��CT) 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 ��Ct values from the training set.