Stability of fecal miRNAs.

<p>To evaluate the long-term stability of the samples, we performed miRNA analyses in the fecal samples collected at different time points. Samples 1–15 were collected between 2004 and 2006 and samples 16–30 between 2009 and 2010 from healthy subjects. All samples were stored and processed in similar conditions. Figure (A) shows variations in miR-16 and miR-196a expression among all feces samples. (B) miR-16 and (C) miR-196a expression in subgroup analyses showed similar expression (p>0.1). (D) Normalized miR-196a expression is comparable in long- and short-term stored samples (p = 0.441). (E) Since miR-216a was present in feces at lowest concentrations, and its expression was analyzed by two independent quantitative RT-PCR runs to evaluate the reproducibility of the analysis (p<0.0001). Normalization was performed with using miR-16 as internal normalizer.</p

Feasibility of Fecal MicroRNAs as Novel Biomarkers for Pancreatic Cancer

<div><h3>Introduction</h3><p>Pancreatic cancer (PCA) is an aggressive tumor that associates with high mortality rates. Majority of PCA patients are diagnosed usually at late tumor stages when the therapeutic options are limited. MicroRNAs (miRNA) are involved in tumor development and are commonly dysregulated in PCA. As a proof-of-principle study, we aimed to evaluate the potential of fecal miRNAs as biomarkers for pancreatic cancer.</p> <h3>Materials and Methods</h3><p>Total RNA was extracted from feces using Qiagen's miRNA Mini Kit. For miRNA expression analyses we selected a subset of 7 miRNAs that are frequently dysregulated in PCA (miR-21, -143, -155, -196a, -210, -216a, -375). Subsequently, expression levels of these miRNAs were determined in fecal samples from controls (n = 15), chronic pancreatitis (n = 15) and PCA patients (n = 15) using quantitative TaqMan-PCR assays.</p> <h3>Results</h3><p>All selected miRNAs were detectable in fecal samples with high reproducibility. Four of seven miRNAs (miR-216a, -196a, -143 und -155) were detected at lower concentrations in feces of PCA patients when compared to controls (p<0.05). Analysis of fecal miRNA expression in controls and patients with chronic pancreatitis and PCA revealed that the expression of miR-216a, -196a, -143 und -155 were highest in controls and lowest in PCA. The expression of the remaining three miRNAs (miR-21, -210 and -375) remained unchanged among controls and the patients with either chronic pancreatitis or PCA.</p> <h3>Conclusion</h3><p>Our data provide novel evidence for the differential expression of miRNAs in feces of patients with PCA. If successfully validated in large-scale prospective studies, the fecal miRNA biomarkers may offer novel tools for PCA screening research.</p> </div

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Clinico-pathological characteristics of patients included to the study.

*<p> <i>- at time point of sample collection;</i></p>#<p> <i>- endocrine pancreatic insufficiency is defined by impaired glucose tolerance test or by manifest diabetes mellitus;</i></p>§<p>- exocrine pancreatic insufficiency is defined by reduced elastase in stool.</p

Cumulative miRNA expression analyses improve the separation of PCA samples.

<p>Summation of the ΔCt-values of miR-196a, -216a, -143 and -155 was performed to calculate the Σ ΔCt-value. *- p<0.05, ***- p<0.001. Abbreviations: N-control subjects, CP- chronic pancreatitis, PCA- pancreatic cancer. The data are present as box-and-whiskers plots: the upper and lower limits of the boxes indicate the 75^th and 25^th percentiles, the lines inside the boxes - the medians, and the upper and lower horizontal bars denote the 90^th and 10^th percentiles, respectively.</p

The expression of analyzed fecal microRNAs in comparison to tumor tissues, pancreatic fluid and blood of patients with pancreatic cancer.

<p>The expression of analyzed fecal microRNAs in comparison to tumor tissues, pancreatic fluid and blood of patients with pancreatic cancer.</p

Detection of fecal miRNAs that are commonly dysregulated in pancreatic cancer tissues.

<p>(A) MiRNA microarray expression analyses were performed using Illumina microarray to evaluate the presence of selected miRNAs (miR-16, -375, -196a, -216a, -21, -143, -155 and -210) in a single stool sample of the healthy subject. Fecal miRNA expression was converted to log-expression values following Lumi Bioconductor normalization. (B & C) Expression of selected miRNA was confirmed in all 45 samples including controls, chronic pancreatitis and pancreatic cancer patients. Figure (B) represents the raw miRNA expression Ct-values for qualitative assessment. (C) Normalization was performed using standard ΔCt-method using miR-16 as internal fecal normalizer.</p

MiRNA expression patterns in patients with chronic pancreatitis and PCA.

<p>Figures (A) to (G) represent different miRNAs that were selected for the study based on the alterations in PCA tissues. *represents p<0.05, ***- p<0.001. Abbreviations: N-control subjects, CP- chronic pancreatitis, PCA- pancreatic cancer. The data are present as box-and-whiskers plots: the upper and lower limits of the boxes indicate the 75^th and 25^th percentiles, the lines inside the boxes - the medians, and the upper and lower horizontal bars denote the 90^th and 10^th percentiles, respectively.</p

JCV miR-J1-5p detection in feces.

<p>(A) To test whether JCV miRNA is present in stool, we extracted total RNA from stool samples and performed TaqMan based miRNA expression analyses. Expression of miR-J1-5p was normalized to mean miR-16 and -26b levels and further adjusted to the sample with the lowest miR-J1-5p expression level (1*). (B) To test the reproducibility of miRNA detection, we performed independent RNA extraction from the same samples in the subset of fecal samples from healthy subjects (n = 5). The samples were normalized to mean miR-16 and -26b expression. (C) Concomitant expression analyses of miR-J1-5p and -3p showed no correlation with JCV miRNA expression, arguing for potential cross-reactivity with BKV microRNA. (D&E) To measure JCV miR-J1-5p expression in feces from CRC patients, miR-J1-5p was analyzed by TaqMan PCR in 29 FOBT specimens from patients without and with colorectal neoplasia. Fold-expression was calculated using the 2^−ΔCt method normalized to mean miR-16 and -26b expression. D Represents the single sample values and E the mean values ± SD.</p

JCV miR-J1-5p detection in CRC patient tissues.

<p>(A) Six FFPE tissue specimens were stained for JCV T-Ag expression to ensure the active presence of JCV in CRC tissues. Figure A1 shows a representative image for strong, and A2 shows weak JCV-T-Ag protein expression. (B) JCV miR-J1-5p expression was evaluated in each of 3 samples with strong and weak JCV T-Ag expression. Normalization of miR-J1-5p expression in FFPE tissues was performed using miR-16, as previously validated. (C & D) miR-J1-5p expression was evaluated in paired normal colonic mucosa and CRC fresh frozen tissues from 21 patients with CRC. In C, miR-J1-5p expression is shown for paired normal colonic mucosa and CRC tissues. miRNA expression is shown as 2^−ΔCt normalized to RNU6b expression. (E) miR-J1-5p expression in CRC tissues is shown correlated with miR-J1-5p expression in normal colon mucosa. The results are presented as 2^−ΔΔCt normalized to RNU6b and matching normal colonic mucosa, and the values are sorted in descending order. From a total of 21 CRC tissues samples, 12 samples (below the line) showed lower, and 6 samples (above the line) higher miR-J1-5p expression in CRC tissues compared to normal mucosa.</p