Immediately following CO2 asphyxiation, rats were perfused though the heart with warm saline. The cervix was excised, carefully trimmed, rinsed (1 ml of 0.1M phosphate buffered saline), minced with scissors, and cells dispersed (1mg/ml Collagenase B with DNAase in 25 mM HEPES-HBSS at 37° C with agitation by pipette every 0.5 h for 1.5 h). The suspension was passed through a 70 μm filter, centrifuged, washed and, recentrifuged (10 min at 1200 rpm at 4° C), before the cell pellet was resuspended in 2 ml HEPES-HBSS. Isolated cervix cells from each of 3 individual rats in the NP and D21 groups were flash frozen at -80C in liquid nitrogen and stored for microchip analysis of gene expression. Cells from the cervix of the remaining 3 rats in each group were subjected to a magnetic bead separation procedure to remove Mφs. Briefly, a 5 µl aliquot of the final cell suspension was mixed with 5 µl trypan blue and cell counts determined with a hematocytometer. An aliquot of the Mφ-specific ED1 antibody (1µg/106 cells in 0.1 ml; Serotec, Oxford, UK) was added to the cervix cell suspension. The suspension was incubated for 10 min at 8° C, centrifuged, then the cell pellet washed twice in HEPES-HBSS (2 ml/107 cells). As per instruction, the cell pellet was resuspended (about 107cells/ml at 8° C) and a 0.025 ml aliquot of Dynabeads in HEPES-HBSS was added (0.1ml of beads solution/106 cells; DynaMag, Invitrogen Inc). After 30 min of gently rotation and agitation at 10 min intervals, each vial was placed next to a magnet apparatus for 2 min to quarantine ED-1 bound Mφs. The Mφ-depleted supernatant was decanted into a microcentrifuge tube, flash frozen, and store at -80° C for gene expression analyses.
Extracted molecule
total RNA
Extraction protocol
Samples were lysed in Tri-reagent and total RNA isolated using phenol/chloroform extraction followed by purification over spin columns (reagents from Ambion, Austin, TX). The concentration and purity of total RNA was measured by spectrophotometry at OD260/280 and the quality of the total RNA sample was assessed using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip (Agilent Technologies, Santa Clara, CA)
Label
Cy3
Label protocol
Labeled cRNA was prepared by linear amplification of the Poly(A)+RNA population within the total RNA sample. Briefly, 1 µg of total RNA was reverse transcribed after priming with a DNA oligonucleotide containing the T7 RNA polymerase promoter 5’ to a d(T)24 sequence. After second-strand cDNA synthesis and purification of double-stranded cDNA, in vitro transcription was performed using T7 RNA polymerase. The quantity and quality of the labeled cRNA was assayed by spectrophotometry and the Agilent Bioanalyzer.
Hybridization protocol
1 µg of the purified cRNA from each individual cervix was fragmented to uniform size and applied to the Rat Gene Expression 4x44K v3 microarray chips in hybridization buffer (Agilent Technologies). Arrays were hybridized at 65° C for 17h in a shaking incubator and washed at 37° C for 1 min.
Scan protocol
Rinsed and dried arrays were scanned at 5 µm resolution with an Agilent G2565 Microarray Scanner (Agilent Technologies)
Description
Gene expression in the non-pregnant cervix not due to macrophages
Data processing
Data from scanned images of arrays were processed and analyzed with Feature Extraction and GeneSpring GX v7.3.1 software (Agilent Technologies).
