MicroRNA Expression in Stepwise Processed Plasma Samples Using qRT-PCR Profiling.

<p><i>(A)</i> Heat map of relative expression (in CTs) of 282 detected miRNAs assayed in parallel using the Exiqon qRT-PCR array. Colors represent the greatest relative expression differences across the 5 different processing conditions for each individual miRNA. The processing condition(s) with the highest relative expression is shown in red and lowest in green for each miRNA. miRNAs are arranged left to right in order of decreasing difference between CTs across all 5 samples. Selected miRNAs are highlighted above. Ranges of average fold-differences are indicated along the bottom, where fold-differences are calculated as = 2^(ΔCT). <i>(B)</i> Validation of selected miRNAs by individual qRT-PCR assay. MicroRNAs are arranged in order of decreasing range between highest and lowest fold difference. Numbers in each box represent the normalized CT difference from the mean of all CT values for each miRNA evaluated in sample type. Color schema follows panel A. Average miRNA expression fold-differences between Plasma_RICH vs. Plasma_POOR, Plasma_STD vs. Plasma_FILT, and Plasma_POOR vs. Plasma_FILT is indicated in the bottom rows. Fold-differences are calculated as = 2^(ΔCT).</p

MicroRNA Biomarker Study Comparing Plasma from Lung Cancer Cases and Controls.

<p><i>(A)</i> Average miRNA cycle threshold (CT) of plasma from lung cancer cases on the x-axis and from controls on the y-axis. Dotted line represents the line of identity. <i>(B)</i> Bar graph of average particle counts (thousand/µL) in the platelet and microparticle size ranges of plasma from lung cancer cases and controls. Student's t-test, two-tailed, <i>p = </i>0.02 and 0.13, respectively. Error bars represent standard deviation.</p

Serum Processing and Associated Particle Content.

<p><i>(A) Left</i>: Schematic of Serum_STD processed with a 0.22 um filtration step to form Serum_FILT. <i>Right:</i> Histogram content of platelets and microparticles in stepwise processed plasma samples. X-axis is particle diameter and Y-axis is number of particles/mL. (B) Platelet and microparticle content (in thousand/µL) of serum samples as measured by particle counter and hematology analyzer. <i>(B)</i> Validation of selected miRNAs by individual qRT-PCR assay. Normalized CT difference from mean of all 7 samples (including 5 plasma samples) is shown. MicroRNAs are arranged as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064795#pone-0064795-g003" target="_blank">Figure 3</a>. Average fold difference between Serum_STD vs. Serum_FILT is indicated below.</p