Article Figures & Data
Figures
- Figure 1.
qPCR measurements of sources of blood miRNA variability. A, proportions of interindividual, intraindividual, and technical variability were estimated for 12 subjects using a mixed-effects model of qPCR data from seven target miRNAs (miR16, miR342-3p, miR30d, miR185, let7d, miR130a, miR451), two endogenous control small RNAs (RNU48 and snRNA U6), and one exogenous spike-in (cel-39). Technical variability includes variability within- and between-extraction batches as well as plate-to-plate variability. B, interclass correlation (ICC) for each source of variability was calculated as the proportion of total variance for each miRNA.
- Figure 2.
Cumulative distributions of minimum detectable fold changes in miRNAs for bootstrap procedure using repeated measures. Smallest fold changes detected for the 143 miRNAs (with 80% statistical power) are plotted under the seven experimental design conditions (for N = 75 vs. 75 subjects), which vary in proportion of repeated measures [(A) 20%, (B) 50%, and (C) 100%] and number of repeated measures per subject (n1 = 0, n1 = 1 or n1 = 4). Fold changes are reported with 80% power for simulations with 100 bootstraps for 4 unique subjects each with 3 time point measurements (25). The vertical line in each figure is for purposes of comparing distributions at a 2-fold change in miRNA expression.
- Figure 3.
Comparison of 50% repeated measure designs for detection of significant fold changes in miRNAs. Designs 0, 2A, and 2B were compared with each other to calculate the proportion of the 143 miRNAs for which two designs' CIs do not overlap at a given P value. (Designs differ by number of repeated measures for each subject.)
Tables
- Table 1.
Summary table of experimental designs used for simulations of miRNA microarray data
Design N1 N2 Number of subjects with repeated measures (%) Number of repeated measures 0 75 75 0 (0) 0 1A 75 75 30 (20) 1 1B 75 75 30 (20) 4 2A 75 75 75 (50) 1 2B 75 75 75 (50) 4 3A 75 75 150 (100) 1 3B 75 75 150 (100) 4 - Table 2.
Variance terms and P values for sources of variability
Random effect qPCR replicate Interindividual Seasonal Between batches Within batch Residual miRNA Target Variance P value Variance P value Variance P value Variance P value Variance P value Variance Total miR130a 0.826 <0.001 0.149 0.387 0.000 1.000 0.000 1.000 0.000 1.000 0.089 1.064 miR30d 0.541 <0.001 0.053 0.560 0.000 1.000 0.000 1.000 0.000 1.000 0.100 0.693 miR185 0.820 <0.001 0.222 0.153 0.031 0.910 0.000 1.000 0.149 1.000 0.102 1.324 let-7d 0.441 <0.001 0.234 0.103 0.000 1.000 0.036 0.770 0.271 0.647 0.040 1.021 miR16 (TaqMan) 0.499 <0.001 0.237 <0.05 0.045 0.706 0.036 0.888 0.000 1.000 0.113 0.929 c.eleg-39 0.137 <0.001 0.000 1.000 0.000 1.000 0.011 0.815 0.000 1.000 0.080 0.228 RNU48 0.388 <0.001 0.097 0.264 0.000 1.000 0.000 0.213 0.000 1.000 0.086 0.571 SNU6 0.375 <0.001 0.000 1.000 0.000 1.000 0.000 1.000 0.000 1.000 0.458 0.833 miR16 (qScript) 0.119 <0.001 0.129 <0.001 0.000 1.000 0.000 1.000 0.346 <0.05 0.014 0.607 miR451 0.308 <0.001 0.386 <0.001 0.401 <0.05 0.029 0.694 0.482 0.073 0.034 1.640 miR342-3p 0.164 <0.001 0.009 0.535 0.000 1.000 0.006 1.000 0.058 1.000 0.033 0.271 NOTE: The variability for each random-effect term in the model is reported, as well as P values based on ANOVA tests for each term of each modeled miRNA.
- Table 3.
Estimations of minimum detectable fold changes for 2 study designs
miR451 miR185 Study 1 Study 2 Study 1 Study 2 Mean 3.77 2.38 4.18 2.4 SE 0.95 0.53 1.2 0.67 95% CI (1.75–5.16) (0.97–3.36) (2.67–6.46) (1.71–3.74) 90% CI (1.88–5.11) (1.07–3.20) (3.69–6.37) (2.11–3.68) NOTE: The estimates of variability obtained from the empirical qPCR study of miR451 and miR185 were used to determine the minimum detectable fold change with 80% statistical power for a theoretical study (N = 75 vs. 75) of two miRNAs. The mean fold change, SE, 95% CI, and 90% CI are reported given no replicates (Study 1) versus a study given five extraction batches, five within-batch replicates, five time point replicates, and five qPCR replicates for 50% of the subjects (Study 2).
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - Supplementary Methods for miR Extraction, qPCR, and statistical analysis
- Supplementary Results - Supplementary Results for Figures S1-S4
- Supplementary Legends - Supplementary Legends for Figures S1-S4
- Supplementary Figure S1 - Supplementary Figure S1A and S1B: miR Extraction Methods Comparison
- Supplementary Figure S2 - Supplementary Figure S2A and S2B: miR qPCR Assay Comparison
- Supplementary Figure S3 - Supplementary Figure S3A-S3C: Cumulative Distributions including 95% Confidence Intervals
- Supplementary Figure S4 - Supplementary Figure S4A and S4B: Comparison of Designs 0/1A/1B and Designs 0/3A/3B
Volume 23, Issue 12
