Fig 1.
Evaluation of Cq values of UniSp2, miR-103a-3p and miR-451a in four samples using different isolation protocols and RNA carriers. y, yeast RNA carrier; m, MS2 RNA carrier; w, without carrier; Q, miRNeasy Mini kit modified protocol; E, miRCURY RNA isolation kit Biofluids modified protocol. Mean and standard deviation values are indicated under each box plot diagram.
Table 1.
Main characteristics of the miRNAs analyzed by qRT-PCR.
Fig 2.
Correlations between mean “fold recovery” (FR) values of miRNAs and GC content of each miRNA, using yE protocol (A) and yQ protocol (B). yE, miRCURY RNA isolation kit Biofluids modified protocol using yeast RNA carrier; yQ, miRNeasy Mini kit modified protocol using yeast RNA carrier; FR, the fold recovery of each miRNA vs the same protocol without carrier.
Fig 3.
Correlations between mean “fold recovery” (FR) values of miRNAs and base 10 logarithm of miRNA relative abundance in sample (SRA), using yE protocol (A) and yQ protocol (B). yE, miRCURY RNA isolation kit Biofluids modified protocol using yeast RNA carrier; yQ, miRNeasy Mini kit modified protocol using yeast RNA carrier; FR, the fold recovery of each miRNA vs the same protocol without carrier; SRA, the mean relative abundance of each miRNA on samples isolated without carrier.
Fig 4.
Correlations between mean “fold recovery” (FR) values of miRNAs and ΔG of each miRNA, using yE protocol (A) and yQ protocol (B). To avoid the influence of GC content in the analysis of ΔG, for these correlations we used only miRNAs with a GC content between 25% and 75% percentile. yE, miRCURY RNA isolation kit Biofluids modified protocol using yeast RNA carrier; yQ, miRNeasy Mini kit modified protocol using yeast RNA carrier; FR, the fold recovery of each miRNA vs the same protocol without carrier; ΔG, the free energy of the most stable secondary structure of each miRNA.
Fig 5.
miRNA levels normalized by plasma volume and by an endogenous miRNA.
Fold abundance of miRNAs normalized by plasma volume (FR of miRNAs, left column) and normalized by hsa-miR-103a-3p (apFC of miRNAs, right column) and referred to control plasma samples isolated with the E protocol without carrier (in gray). Data were determined in plasma RNA-derived samples isolated with Q and E modified protocols with yeast RNA as carrier (y) or without RNA carrier (w). E, miRCURY RNA isolation kit Biofluids modified protocol; Q, miRNeasy Mini kit modified protocol; FR, fold recovery of miRNAs vs wE protocol; apFC, apparent fold change of miRNAs vs wE protocol. P-values were calculated using Wilcoxon Signed Rank Test, Exact Signification two-tailed, with the IBM SPSS Statistics 20 software. ϕP<0.05, and ϕϕP<0.001 vs wQ; €P<0.05, and €€P<0.001 vs yE; ψP<0.05, and ψψP = 0.001 vs wE. Mean and standard deviation values are indicated under each box plot diagram.