Hierarchical clustering of microarray data (Spearman).

<p>Cancer samples and normal donor samples (brackets) were clustered using a hierarchical clustering program to show sample-to-sample relationships. Sample labels include donor condition (cancer type or normal), sample lot number (last three digits), gender, and cancer stage (2 – 4, or 0 for normal). Labels marked with a or b indicate repeat testing of the same sample.</p

Summary of normal donor serum samples tested in this study.

<p>Summary of normal donor serum samples tested in this study.</p

Summary of human cancer serum samples tested in this study.

<p>Summary of human cancer serum samples tested in this study.</p

Assay sensitivity.

<p>RNA miRNA analog oligonucleotides, at concentrations ranging from 0 to 40 million copies per microliter, were spiked into 400 ul of serum after the addition of RLT buffer. RNA was then extracted from the serum using phenol/chloroform extractions and an ethanol precipitation. Samples were then labeled and hybridized on a microarray. Vertical bars indicate array signal intensities for specific miRNA probes representing the wild type sequence (Wild) and probes with two internal mutations (mut) for (A) oar|miR-431 and (B) oar|miR-127. Scales for the 4,000 and 0 copies data points (boxed in left panels) are expanded in the right panels: (C) oar|miR-431, and (D) oar|miR-127.</p

Heat maps of microRNA array data.

<p>The set of miRNAs used for analysis was chosen based on significance in at least 5 hybridizations. A probe-set was judged significant if the ratio of perfect-match (PM)/mismatch (MM) probes was greater than 1.5.For each hybridization in the analysis, only significant signal was used for the clustering. Signal for those miRNAs whose signal was judged significant by PM/MM ratios was Log 2 converted. Then the signal was median normalized over that hybridization and Average Linkage Clustering was performed using a Spearman Rank Correlation. Clustering was visualized using the program TreeView <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006229#pone.0006229-Eisen1" target="_blank">[12]</a>. Green indicates negative values and Red indicates positive values. Samples are identified as either normal (blue bar) or cancer (yellow bar) at the top of the figure. miRNA names are listed to the right and sample identifications are listed above.</p

Up-regulation of cancer sera miRNAs over normal donor sera miRNAs.

<p>Log transformed normal donor serum miRNA signals (blue line) were compared to miRNA array signals from a prostate cancer cell line 22Rv (open squares) and from a prostate cancer patient (closed diamonds). In general, cancer and cell line miRNAs seem to be up-regulated when compared to normal donor serum miRNAs.</p

Probe design scheme for miRNA array.

<p>Sequences for miRNA probes were taken from the Sanger database version 10.0 (released, 8/2/2007). We generally used only the predominantly expressed form for each miRNA precursor. This was done to save space on the array. The final list included all of the dominant miRNA forms from the studies referenced in this paper <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006229#pone.0006229-Lu1" target="_blank">[4]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006229#pone.0006229-Resnick1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006229#pone.0006229-Rosenfeld1" target="_blank">[18]</a>. Three probes were designed for each mature miRNA sequence: 1) antisense to wild type; 2) double mutated antisense (boxes); and 3) sense negative control probes (included if the corresponding sequence was not found in miRNA databases). Hsa-let-7a is used here as an example of our approach to probe design.</p

Analysis of microRNA data for normal and prostate cancer sera.

<p>After data set normalization, the natural log of the ratio of the signal for a specific probe over the same probe from the normal serum sample was taken. 15 miRNAs showed up-regulation in all stage 3 and 4 prostate cancer samples when compared to sera from normal male donors. These miRNAs are listed below each data set. Five stage 3 and 4 prostate cancer sera (Yellow), and 8 normal male donor sera (red) were analyzed. Vertical lines indicate plus or minus one standard deviation of the mean.</p

EGA Reation Scheme

<p>EGA Reation Scheme</p

The design of the linker used in this experiment

<p>The design of the linker used in this experiment</p