For the purpose of the study, two pieces of placental tissue approximately 1×1×1cm in size were randomly obtained from the central part of the placenta maternal phase, as previously described. Tissues were washed with isotonic saline solution to remove excess of blood cells and immediately stored at -80 °Co until further processing.
Extracted molecule
total RNA
Extraction protocol
For total RNA extraction, 100mg of placental tissue were mechanically homogenized in TRIzol (Life Technologies, Foster City, CA), treated with DNase I (Ambion, Austin, USA) and enriched for small RNAs (siRNAs, miRNAs) using the mirVana microRNA Isolation kit (Ambion, Austin, USA). RNA quantity and purity were measured using a Nanodrop spectrophotometer (ND-1000, NanoDrop Technologies, Houston, TX, USA).
Label
Cy5
Label protocol
Labeling and hybridization were performed using the LabelIT miRNA labeling kit (Mirus Bio LLC, USA) according to the manufacturer’s instructions. Samples were hybridized to an Applied MicroArrays (miRlink Bioarray 300054-3PK) platform containing 1211 human miRNAs (Applied Biosystems, Foster City, CA).
Hybridization protocol
Slides were hybridized using the manufacturers protocol (APPLIED MICROARRAYS miRLinkTM v16 Bioarray Protocol).
Scan protocol
Images were scanned using an Agilent Microarray Scanner (G2565CA).
Description
Raw_Data_Slide_3.txt, Block8
Data processing
Microarray raw data were extracted, as previously described, using the Imagene 6.0 software (Biodiscovery Inc., USA). The MATLAB® simulation environment (The Mathworks, Inc, Natick, MA) was applied for multi-parameter analyses. Normalization was performed using the quantile normalization algorithm. The two tailed student t-test was used to assess the mean differences between the two groups. Relative expression was estimated as the log2-transformed ratio of the individual miRNA expression levels over the mean expression level of all control samples for the respective miRNA under investigation. Final miRNA values have been calculated as the mean of each specific miRNA replicates on each array. Continuous variables were expressed as median ± standard deviation, unless differently indicated. MiRNAs were considered significant if they had a p-value<0.05 and False Discovery Ratedetection Rate (FDR) ≤ 0.05.
