All samples were fractionated and milk extracted immediately.
Growth protocol
Milk samples were taken under sterilization conditions using pumps and sterile bottles, shields, etc. Blood was taken into EDTA to obtain plasma and PBMCs.
Extracted molecule
total RNA
Extraction protocol
All cells and macromolecules were lysed, and then washed three times using different reagents, then resoluble using RNase free water. These four miRNA extraction kits are all filter column-based methods. All the extractions were done according to the manufacturer’s protocol except the amount of lipid and lysis reagent in the miRCURY RNA Isolation-Biofluids Kit, where 400 μL of lipid in 120 μL of lysis reagent was used.
Label
FAM
Label protocol
Reverse transcription was performed from 200 ng total RNA using RT kit (life technologies), megaplex RT primer pool was used. Cycler parameters for RT reaction was 40 cycles of 16C for 2 min, 42C for 1 min, and 50C for 1 sec. Then 85C hold for 5 min. All cDNA were preamplified using Taqman PreAmp master mix and megaplex PreAmp Primers. Cycler parameters for PreAmp reaction, 95C for 10 min, 55C for 2 min, 72C for 2 min, 12 cycles of 95C for 15 sec and 60C for 4 min. then hold at 99.9C for 10 min. All samples were applied into 14 OpenArray panels (3 samples per one panel) suing openarray loader. Then OpenArray real time PCR cycler was used to run all panels.
Hybridization protocol
n/a
Scan protocol
n/a
Description
Maternal_blood
Data processing
The HTqPCR software was provides biostatistical methods for the analysis of Ct values from TaqMan OpenArray (OA) assays. The software package handles data loading, quality assessment, normalisation, visualisation and parametric or non-parametric testing for statistical significance in Ct values between features (the individual microRNAs). For the normalisation, RNU48, RNU44 and U6 rRNA as endogenous controls (miRNA) were used. The primary experimental data were loaded into the software according to the authors’ recommendations, where Ct≤29 is considered as detectable microRNA. Also, in each sample group, miRNAs that was identified in 4 or more samples of each group with Ct value between 5 and 29 were considered as reliable microRNAs. Highly expressed miRNAs were also determined at more strict conditions, where the miRNA Ct value must have been between 8 and 12, and present in at least 4 samples in each sample group. Complete raw data were normalised using the deltaCt method, which scales the individual samples while retaining the distribution of Ct values. Normalised miRNAs were done in R without generating deltaCT values, thus only the list of normalized miRNA with Ct vlaue between 8 and 29 and that detected in at least 4 samples out of 10 analysed in each group is provided ('normalized_miRNAs_list.txt'). The 'fold_change.txt' contains only differentially expressed miRNAs between milk lipid and milk cell samples.