Mathematical Details from Effect of aspirin on tumour cell colony formation and evolution

Mathematical Detail

Clinico-pathological characteristics of patients and healthy volunteers in the study.

<p>*Clinical characteristics of one patient was incomplete.</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 miRNA sequence and detection.

<p>(A) Schematic presentation of the JCV genome. The black circle marks the transcript location of the JCV miR-J1 stem loop. (B) JCV miR-J1-5p and -3p sequences are compared to the Merkel Cell Polyomavirus-, SV40- and BK virus-miRNA sequences. (C & D) CRC cells were transfected in vitro with a JCVT-Ag-E plasmid, and JCV T-Ag message and miRNA expression were analyzed. In C, GAPDH and β-actin were used as loading controls for mRNA and protein expression, respectively. (D) Vector transfected cells showed no detectable miR-J1-5p expression, while JCV miR-J1-5p expression was high in transfected cells. To measure the expression of miR-J1-5p, expression in the vector was set to a Ct-value of 40, and 2^−ΔΔCt values were calculated using RNU6b for normalization.</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

Technical Factors Involved in the Measurement of Circulating MicroRNA Biomarkers for the Detection of Colorectal Neoplasia

<div><p>Background</p><p>Circulating miRNAs are emerging as promising blood-based biomarkers for colorectal and other human cancers; however, technical factors that confound the development of these assays remain poorly understood and present a clinical challenge. The aim of this study was to systematically evaluate the effects of factors that may interfere with the accurate measurement of circulating miRNAs for clinical purposes.</p><p>Methods</p><p>Blood samples from 53 subjects, including routinely drawn serum samples, matched plasma from 30 subjects, and matched serum samples drawn before and after bowel preparation for colonoscopy from 29 subjects were collected. Additionally, 38 serum specimens stored in the clinical laboratory for seven days were used to test the stability of miRNAs. Hemolysis controls with serial dilutions of hemoglobin were prepared. RNA was extracted from serum, plasma or hemolyzed controls with spiked-in cel-miR-39, and levels of miR-21, miR-29a, miR-125b and miR-16 were examined by real-time RT-PCR. Hemolysis was measured by spectrophotometry.</p><p>Results</p><p>The expression levels of miR-16 and the degree of hemolysis were significantly higher in plasma than in serum (P<0.0001). Measured miR-21, miR-29a, miR-125b and miR-16 expression increased with hemoglobin levels in hemolyzed controls. The degree of hemolysis in serum samples correlated significantly with the levels of miR-21 (P<0.0001), miR-29a (P = 0.0002), miR-125b (P<0.0001) and miR-16 (P<0.0001). All four miRNAs showed significantly lower levels in sera that had been stored at 4°C for seven days (P<0.0001). Levels of miR-21 (P<0.0001), miR-29a (P<0.0001) and miR-16 (P = 0.0003), and the degree of hemolysis (P = 0.0002) were significantly higher in sera drawn after vs. before bowel preparation.</p><p>Conclusions</p><p>The measured levels of miRNAs in serum and plasma from same patients varied in the presence of hemolysis, and since hemolysis and other factors affected miRNA expression, it is important to consider these confounders while developing miRNA-based diagnostic assays.</p></div

MiR-21, miR-29a, miR-125b and miR-16 in serial dilutions of hemolyzed control samples.

<p><b>A:</b> Image of serial dilution of hemolyzed control samples. <b>B:</b> Correlation between dilutions of hemolyzed control samples and the degree of hemolysis is shown (Pearson’s correlation coefficient = R²). <b>C:</b> Levels of miR-21, miR-29a, miR-125b and miR-16 were elevated from baseline in hemolyzed control with 1/64^th dilution, and their levels increased along with the presumed hemoglobin concentration.</p

The degree of hemolysis in human serum samples.

<p><b>A:</b> The relationship between the visual hemolysis score and the degree of hemolysis which was determined by spectrophotometry. The hemolysis score of each serum sample was visually designated as follows: 0, no sign of hemolysis; 1, slight hemolysis cannot be ruled out because of dark yellow discoloring; 2, hemolysis is strongly suspected by orange to pink discoloring; 3, evident hemolysis with dark pink to red discoloring. <b>B:</b> Correlation between the degree of hemolysis and serum levels of miR-21, miR-29a, miR-125b and miR-16. The Spearman’s rank correlation coefficient (ρ) is shown. <b>C:</b> Correlation between the degree of hemolysis and serum levels of miR-21, miR-29a, miR-125b and miR-16 in visually non-hemolysed sera (hemolysis score of 0 and 1). The Spearman’s rank correlation coefficient (ρ) is presented.</p

Measured miRNAs in matched serum and plasma.

<p>Expression levels of miR-21, miR-29a, miR-125b and miR-16 (<b>A</b>) and the degree of hemolysis (<b>B</b>) are illustrated. Absorbance of each serum sample at 560 nm, 576 nm and 592 nm was measured by spectrophotometry and the degree of hemolysis was calculated by the following formula: estimated hemoglobin level = 2*OD^{576 nm}−OD^{560 nm}−OD^{592 nm}. Differences between serum and plasma were analyzed by the Wilcoxon signed-rank test.</p

Study subjects and blood samples used in this study.

<p>The chart provides the description of patients and healthy volunteers from whom blood samples were collected under various conditions during the course of this study.</p