Comparison of qRT-PCR controls obtained from RNA with and without treatment with Bacteroides heparinase I. Panel A: Workflow of conditions tested for serum and plasma. Serum under standard conditions and plasma treated with Bacteroides heparinase I during the reverse transcription (RT) of RNA to cDNA yielded the most consistent signals. The colors indicate the level of performance – poor (red); fair (yellow); good (green). Panel B. Comparison of Ct values for qPCR controls obtained from RNA with and without treatment with Bacteroides heparinase I. Positive PCR Control (PPC) denoted a positive qPCR control and miRTC denotes reverse transcriptase controls. The plasma was spiked with C. elegans miR-39. Results (duplicate readings) for replicate samples for each treatment are shown. Optimization steps (Experiments 1 and 2) were carried out with plasma donated by the same persons. [Selected treatment conditions were confirmed using additional plasma (Figure 3, Additional file 1: Figure S1).] Panels C-E, Threshold cycles (Ct ) for qPCR controls with and without heparinase I. miRTC (panel C), PPC (D), and miR-cel-39 (E) curves of Relative Fluorescent Units RFU (Y-axis) versus cycles (X-axis) for (left to right): 1) 6 U Bacteroides heparinase I during RT, 2) 6 U Bacteroides heparinase I treatment prior to RT, 3) 0.6 U Bacteroides heparinase I before RT, and 4) no treatment in duplicate (not visible along X-axis in Panel C, E). Panels F-H: miRTC curves of RFU (Y-axis) versus cycle time (X-axis) for (left to right): 1) 12 U, 6 μl; 2) 12 U, 3 μl; 3) 6 U, 3 μl; and 4) 0.6 U, 3 μl for Units of Bacteroides heparinase I and volume of RNA, respectively for miRTC (panel F), PPC (G) and miR-cel-39 (H). Condition four yielded no curve for miR-cel-39 in panel H (presented on X-axis).