Qualitative concordance of detected genes by three sample preparation methods.

<p>Among 24,267 gene spots on the microarray used, genes that were detected in at least 60% of the sample replicates were used to create the Venn diagram.</p

Spearman's rank correlation between the microarray assay and qRT-PCR assay.

<p>UHRR, HBRR, breast, and colon RNA derived from human tissue or cells were processed by the non-amplification (black bar), the 1-round amplification (grey bar), or the 2-round amplification (white bar) method, analyzed by microarray. The obtained PolR2A-normalized signal intensity (log₂) of the 42 genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031397#pone.0031397.s001" target="_blank">Table S1</a>) was compared with ΔCt values of the same samples analyzed by qRT-PCR assays.</p

MicroRNA Markers for the Diagnosis of Pancreatic and Biliary-Tract Cancers

<div><p>It is difficult to detect pancreatic cancer or biliary-tract cancer at an early stage using current diagnostic technology. Utilizing microRNA (miRNA) markers that are stably present in peripheral blood, we aimed to identify pancreatic and biliary-tract cancers in patients. With “3D-Gene”, a highly sensitive microarray, we examined comprehensive miRNA expression profiles in 571 serum samples obtained from healthy patients, patients with pancreatic, biliary-tract, or other digestive cancers, and patients with non-malignant abnormalities in the pancreas or biliary tract. The samples were randomly divided into training and test cohorts, and candidate miRNA markers were independently evaluated. We found 81 miRNAs for pancreatic cancer and 66 miRNAs for biliary-tract cancer that showed statistically different expression compared with healthy controls. Among those markers, 55 miRNAs were common in both the pancreatic and biliary-tract cancer samples. The previously reported miR-125a-3p was one of the common markers; however, it was also expressed in other types of digestive-tract cancers, suggesting that it is not specific to cancer types. In order to discriminate the pancreato-biliary cancers from all other clinical conditions including the healthy controls, non-malignant abnormalities, and other types of cancers, we developed a diagnostic index using expression profiles of the 10 most significant miRNAs. A combination of eight miRNAs (miR-6075, miR-4294, miR-6880-5p, miR-6799-5p, miR-125a-3p, miR-4530, miR-6836-3p, and miR-4476) achieved a sensitivity, specificity, accuracy and AUC of 80.3%, 97.6%, 91.6% and 0.953, respectively. In contrast, CA19-9 and CEA gave sensitivities of 65.6% and 40.0%, specificities of 92.9% and 88.6%, and accuracies of 82.1% and 71.8%, respectively, in the same test cohort. This diagnostic index identified 18/21 operable pancreatic cancers and 38/48 operable biliary-tract cancers in the entire cohort. Our results suggest that the assessment of these miRNA markers is clinically valuable to identify patients with pancreato-biliary cancers who could benefit from surgical intervention.</p></div

Plots of miR-6075 (A) and miR-125a-3p (B) expression signals on a log2 scale in patients with pancreato-biliary cancers, healthy control individuals, and patients with colon, stomach, esophageal, or liver cancer, or with non-malignant abnormalities in either the pancreas or the biliary tract.

<p>Plots of miR-6075 (A) and miR-125a-3p (B) expression signals on a log2 scale in patients with pancreato-biliary cancers, healthy control individuals, and patients with colon, stomach, esophageal, or liver cancer, or with non-malignant abnormalities in either the pancreas or the biliary tract.</p

Expression signals of miR-125a-3p and miR6893-5p that showed the smallest p-values in comparison of pancreatic cancer and healthy control (A), or in comparison of biliary-tract cancer and healthy control (B) in the training cohort.

<p>The error bars indicate standard error. The p-values were Bonferroni-corrected.</p

Plots of p-values in the analysis of pancreatic cancer (P can) (A) or biliary-tract cancer (B can) (B) with healthy control (HC) in z-axis, pancreato-biliary cancer (PB can) with non-malignant abnormalities (NMA) in y-axis, or pancreato-biliary cancer (PC can) with other types of cancer (OTC) in x-axis.

<p>The absolute values of the exponents of p-values on a log₁₀ scale were plotted; therefore, miRNAs that were located in further outside are more statistically significant. The ten miRNA used in the diagnostic indices were specified.</p

Expression signals of validated miRNAs that differentiated pancreato-biliary cancer from non-malignant abnormalities (A), or from cancers of other types (B).

<p>The error bars indicate standard error. The p-values were Bonferroni-corrected.</p

ROC analysis of a combination of four miRNAs (miR-6075, miR-6799-5p, miR-125a-3p, and miR-6836-3p) in a solid line, CEA in a dotted line, and CA19-9 in a slashed line.

<p>The AUC values were 0.949, 0.682 and 0.845 for a combination of the four miRNAs, CEA, and CA19-9, respectively. The analysis was performed in the test cohort.</p

Indexed scores that gave the best discriminant performance in the test cohort with the use of four miRNAs: miR-6075, miR-6799-5p, miR-125a-3p, and miR-6836-3p.

<p>Indexed scores that gave the best discriminant performance in the test cohort with the use of four miRNAs: miR-6075, miR-6799-5p, miR-125a-3p, and miR-6836-3p.</p