Eighty-eight miRNA candidates were generated from a PubMed literature
review with search terms including “miRNA” and “Type I and
Type 2 diabetes, GDM, preeclampsia, adipogenesis, obesity, and nonalcoholic
fatty liver disease” 13 (link),
18 (link)–33 (link) (Supplemental Table 1 ). Individual
candidate miRNA abundance was measured via Multiplex Circulating miRNA assay
(Abcam, FirePlex, Cambridge, MA). Samples were digested and hybridized to miRNA
specific hydrogel particles with a universal biotinylated adapter labeled with a
fluorescent reporter, and quantified with EMD Millipore Guava 8HT flow
cytometer. Positive and negative controls were included to reduce inter-plate
and inter-well variability. MiRNA spike-in target probes measured hybridization
success. Blank hydrogel particles were run to define background fluorescence.
Abcam FirePlex Analysis Workbench software was used for data analysis (https://www.abcam.com/kits/multiplex-immunoassays-firefly-analysis-workbench-software ).
Normalization was performed via geNorm algorithm using the three most stable
miRNAs across all samples (hsa-let-7d-5p, hsa-mir-107, and hsa-mir-342-3p)
34 35 . Data was log converted to
eliminate directional bias. Geometric mean and fold changes were calculated for
each miRNA based on normalized expression data.
review with search terms including “miRNA” and “Type I and
Type 2 diabetes, GDM, preeclampsia, adipogenesis, obesity, and nonalcoholic
fatty liver disease” 13 (link),
18 (link)–33 (link) (
candidate miRNA abundance was measured via Multiplex Circulating miRNA assay
(Abcam, FirePlex, Cambridge, MA). Samples were digested and hybridized to miRNA
specific hydrogel particles with a universal biotinylated adapter labeled with a
fluorescent reporter, and quantified with EMD Millipore Guava 8HT flow
cytometer. Positive and negative controls were included to reduce inter-plate
and inter-well variability. MiRNA spike-in target probes measured hybridization
success. Blank hydrogel particles were run to define background fluorescence.
Abcam FirePlex Analysis Workbench software was used for data analysis (
Normalization was performed via geNorm algorithm using the three most stable
miRNAs across all samples (hsa-let-7d-5p, hsa-mir-107, and hsa-mir-342-3p)
34 35 . Data was log converted to
eliminate directional bias. Geometric mean and fold changes were calculated for
each miRNA based on normalized expression data.