Fig. 2.

Characterization of miRNA stability in human plasma. (A) miRNA levels remain stable when plasma is subjected to prolonged room temperature incubation or freeze-thawed multiple times. (Upper) The graphs show normalized Ct values for the indicated miRNAs measured in parallel aliquots of human plasma samples incubated at room temperature for the indicated times. The experiment was carried out by using plasma from the two different individuals noted. Normalization of raw Ct values across samples is based on the measurement of three nonhuman synthetic miRNAs spiked into each sample at known molar amounts after initial plasma denaturation for RNA isolation (described in detail in SI Text). (Lower) The graphs show normalized Ct values for the indicated miRNAs measured in parallel aliquots of human plasma samples subjected to the indicated number of cycles of freeze-thawing. Raw Ct values were normalized across samples by using the same approach as described above. (B) Exogenously added miRNAs are rapidly degraded in plasma, whereas endogenous miRNAs are stable. Three C. elegans miRNAs (chosen for the absence of sequence similarity to human miRNAs) were chemically synthesized and added either directly to human plasma (from individual 003; described in Table S6) or added after the addition of denaturing solution (containing RNase inhibitors) to the plasma (referred to as “denatured plasma”). RNA was isolated from both plasma samples, and the abundance of each of the three C. elegans miRNAs was measured by TaqMan qRT-PCR (Left), as was that of three endogenous plasma miRNAs (Right). Asterisks indicate that the abundance ratios of cel-miR-39, cel-miR-54, and cel-miR-238 added to human plasma directly, relative to addition to denatured plasma, were 1.7 × 10⁻⁵, 9.1 × 10⁻⁶, and 1.1 × 10⁻⁵, respectively and therefore too low to accurately display on the plot. (C) Abundance of miRNAs in serum and plasma collected from the same individual is highly correlated. Each plot depicts the average Ct values (average of two technical replicates) of the indicated miRNAs measured in serum and plasma samples collected from a given individual at the same blood draw. Results from three different individuals are shown. miRNA measurements were highly correlated in both sample types. Results shown for synthetic C. elegans miRNAs spiked into each plasma or serum sample (after addition of denaturing solution) demonstrate that experimental recovery of miRNAs and robustness of subsequent qRT-PCR is not affected by whether it is plasma or serum that is collected.