Effect of RNA degradation. RNA was extracted from PBL blasts, and RNA degradation was induced by exposure to 90°C for times indicated. (A) Representative picture of RNA electropherogram showing impact of heat exposure on RNA integrity. (B) RQI, as an indicator of RNA quality, was analyzed (x-axis). The numbers 1 to 10 at the top of the graph indicate time points corresponding with the numbers in the RNA electropherogram. mRNA levels were assessed by qPCR, targeting high-abundance (green), medium-abundance (red), and low-abundance (blue) markers. For each marker, the mean PCR signal at t=0 was set to 1. The graph shows means of 4 experiments. *P < .05; #P < .01; **P < .005 vs mean of the first 2 time points. (C) MicroRNA levels were assessed by qPCR, targeting high-abundance (green), medium-abundance (red), and low-abundance (blue) markers. The graph shows means of 3 experiments. **P < .005 vs mean of first 2 time points for SNORD49A. (D-E) PCR signals for individual mRNA transcripts and microRNAs were corrected for reference markers using the 2^-ΔΔCq method. The upper and lower boundaries of the shaded areas indicate maximum and minimum values, respectively. *P < .05 vs first time point. (F-G) Experiments investigating the effect of RNA degradation on PCR signals were repeated with RNA from PBMC obtained directly from blood. mRNA transcripts (F) and microRNAs (G) were analyzed. *P < .05 vs mean of the first 2 time points (F: each mRNA transcript; G: only for SNORD38B and SNORD49A).