participants group: healthy third trimester (gestational week 26-29) pregnant women tissue: placenta gender of baby: male mode of delivery: vaginal gestational age at delivery: 41.6 choline intake for 12 weeks: 480 mg/d
Treatment protocol
Healthy third trimester (gestational week 26-29) pregnant women were randomized to a 12-week choline controlled feeding study. The participants consumed either 480 (n=6) or 930 (n=6) mg choline/d for 12 weeks. The placenta samples were obtained within 90 minutes after the delivery of the babies in the hospital. After removing the amniotic sac, the placenta was visually divided into four quadrants. Full thickness biopsies (0.5cm*0.5cm*placenta depth) were obtained from the center of each quadrant, rinsed with PBS and either flash frozen in liquid nitrogen or immersed in RNAlater (Qiagen, Valencia, CA, USA).
Extracted molecule
total RNA
Extraction protocol
The RNAlater stablized placental samples were homogenized with a homogenizer in 1mL TRIzol® Reagent (Invitrogen, Grand Island, NY) for RNA extraction. Total RNA was purified via a commercially available kit (RNeasy Mini kit, Qiagen, Valencia, CA) with on-column DNase digestion. The concentration and quality of the isolated RNA was assayed with a NanoDrop® ND-1000 instrument (Thermo,Wilmington, DE). For RNA quality, a value higher than 1.8 for the ratio of 260 to 280 was deemed acceptable. The integrity of the RNA samples was further determined using an Agilent 2100 Bioanalyzer (Agilent Technologies,Santa Clara, CA). Only samples of an RNA Integrity Number (RIN) >8.0 were included for microarray.
Label
Cy3
Label protocol
Antisense RNA amplification and cyanine 3 labeling was performed with an Amino Allyl MessageAmp II aRNA amplification kit (Ambion Inc., Grand island, NY) using 1000ng input total RNA according to the manufacturer's instructions. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
Cy3-labelled aRNA 825ng was fragmented at 60°C for 30 minutes in a reaction volume of 55μL containing Agilent fragmentation buffer and Agilent blocking agent following the manufacturers instructions. After fragmentation, the samples were cooled on ice for 1 minute. The 2XGEx Hybridization buffer HI-RPM of 55 μL was added to the fragmented sample mixtures. The sample mixtures prepared above (100μL) was used to hybridized to Agilent Whole Human Gene Expression Microarray 4×44K with an Agilent Microarray Hybridization Chamber (G2534A). Microarray hybridization was performed at 65℃ for 17 hours in the hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
Scan protocol
Slides were scanned on the Agilent Scanner G2505C using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5μm, Dye channel is set to Green and Green PMT is set to 100%).
Data processing
The scanned images were analyzed with Feature Extraction Software 10.5 (Agilent) using protocol GE1_105_Dec08 and Grid: 014850_D_20070820 to obtain background subtracted and spatially detrended Processed Signal intensities. The probesets with signal detected (gBGSubSignal intensity >0) in over 75% of the samples were retained for further processing, otherwise they were excluded from the dataset (which led to data reduction in the processed matrix file). There were 36001 out of the 41108 probesets in the GPL6480 platform that passed the filtering criteria and were retained for further processing. For these 36001 probesets, the gBGSubSignal values were log2 transformed and median normalized to get the final normalized data used for between group analyses.
