Pregnant female mice were euthanized by carbon dioxide asphyxiation followed by cervical dislocation and Folr1-/- and Folr1+/+ embryos collected on gd9.5. Embryos were harvested by caesarian section, placed in ice cold phosphate-buffered saline (PBS) and dissected free from the decidual capsule, chorion, and amnion. Folr1-/- and Folr1+/+ embryos were genotyped by tail PCR. Embryos were examined under a Nikon SMZ1500 stereomicroscope and documented by digital photomicroscopy using a Nikon DXM1200 digital camera (Nikon, Japan).
Growth protocol
Animals were housed by an American Association for Accreditation of Laboratory Animal Care (AAALAC)-approved facility, in a climate-controlled room with a 12-hour alternating light/dark cycle, and maintained on standard Purina Mouse Chow #5015 and water ad libitum. Mature male and female mice were mated overnight. The presence of a vaginal plug the following morning was considered evidence of mating and was designated as gd0. Folr1-/- mice were generated by mating female Folr1+/- mice, maintained on folate supplemented diet for 2 weeks, prior to mating with Folr1+/- sires. Pregnant mice were maintained on this diet throughout gestation. Animal protocols utilized were reviewed and approved by the University of Louisville Institutional Animal Care and Use Committee (IACUC).
Extracted molecule
total RNA
Extraction protocol
Total RNA from embryos was isolated using the Picopure RNA isolation kit (Arcturus, Mountain View, CA) following the manufacturer’s recommendations. Antisense RNA (aRNA) was produced using the RiboAmp OA Amplification Kit (Arcturus) according to the manufacturer’s protocols. The RNA was subjected to one round of first strand cDNA synthesis, followed by second strand cDNA synthesis and in vitro transcription. This was followed by another round of first and second strand cDNA synthesis.
Label
biotin
Label protocol
The double stranded cDNA was transcribed in vitro using the High-Yield RNA Transcript Labeling Kit (Enzo, Farmingdale, NY) according to the manufacturer's instructions using biotinylated CTP and UTP (Affymetrix, Santa Clara, CA). The resultant biotin-labeled cRNA was purified with RNeasy columns (Qiagen) and eluted in 40 µL of RNase-free water. The quality and quantity of biotin-labeled cRNA was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Hybridization protocol
Fifteen µg of labeled cRNA was fragmented in 40 µL of 1X fragmentation buffer (40 mM Tris-acetate pH 8.1, 100 mM K-acetate, 30 mM Mg-acetate) for 35 min at 94°C and assessed by agarose gel electrophoresis. Fragmented cRNA was brought to a total volume of 300 µL with 1X hybridization buffer (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01%Tween 20), 100 µg/mL herring sperm DNA, 500 µg/mL acetylated BSA, 50 pM biotinylated control oligonucleotide B2 and 1X eukaryotic hybridization controls (1.5 pM BioB, 5.0 pM BioC, 25 pM BioD and 100 pM cre; Affymetrix). Each cRNA preparation derived from either Folr1+/+ or Folr1-/- embryos was hybridized to an individual GeneChip® from an identical lot of Affymetrix Mouse Expression Array 430A for 16 hours. GeneChip® arrays were washed and stained using antibody-mediated signal amplification and the Affymetrix Fluidics Station’s standard Eukaryotic GE Wash 2 protocol.
Scan protocol
GeneChips were scanned and the fluorescence intensities were determined using an Affymetrix GeneChip Scanner 3000.
Description
Gene (messenger RNA) expression data from GD-9.5, Folr1+/+ (Wild type) embryos
Data processing
Individual GeneChip® arrays were scanned with the GeneChip® Scanner 3000 (Affymetrix) and images were processed using Affymetrix Microarray Analysis Suite (MAS) 5.0 software, according to Affymetrix protocols. For analysis of microarray data, the GeneChip® images of the gd9.5, Folr1-/- embryo samples were normalized to the corresponding images of the gd9.5, Folr1+/+ embryo samples across all probe pair sets. The full dataset (difference call, fold change, average difference value, and absolute call data) from each of the two KO or three WT samples was obtained using Affymetrix MAS 5.0 and contained expression levels for all 23,000 genes and expressed sequence tags (ESTs), as well as logarithm (base 2) of the estimated fold changes. It is to be noted that although a total of six samples and six GeneChips were used, one chip from Folr1-/- embryos failed quality control tests and did not yield any usable data.
