Background
Methods
Animals and tissue collection
Sample RNA preparation
Microarray analysis
cDNA microarray
Microarray hybridization
Data normalization for microarray
Cluster analysis of microarray data
The search for a transcription factor common to a cluster
Quantitative real-time RT-PCR (QPCR)
Gene | Primer | Sequence | Position |
---|---|---|---|
ANXA1
| Forward | 5' GGCTTTGCTTTCTCTTGCTAAGG 3' | 611–633 |
(NM_175784) | Reverse | 5' TGAATCAGCCAAGTCGTCATTT 3' | 680–669 |
CTDSP2
| Forward | 5' GGCCTGGTGTCCAAGTCCT 3' | 203–221 |
(DT808814) | Reverse | 5' CAGAAAAGGGCCTTGAAGATGT 3' | 267–246 |
MSX1
| Forward | 5' TCCCTTGTTCAGCACCGC 3' | 1207–1224 |
(NM_174798) | Reverse | 5' CGGAGGACAAACCAGAGCA 3' | 1270–1252 |
HSPA1A
| Forward | 5' GCAGACCCGCTATCTCCAAG 3' | 41–60 |
(NM_174550) | Reverse | 5' ACCTGAAAACGGCCCACAG 3' | 117–99 |
HSPA8
| Forward | 5' CAAGCTATGTCGCCTTTACTGA 3' | 115–136 |
(NM_174345) | Reverse | 5' GGATTCATTGCGACTTGGTTC 3' | 188–168 |
SULT1E1
| Forward | 5' CAGGATCATCTGGACAGTGTACCA 3' | 182–205 |
(NM_177488) | Reverse | 5' CCAAGTTTGCCAAAGTAATCTGAA 3' | 259–236 |
TFAP2A
| Forward | 5' CCCAACGAAGTCTTCTGTTCAGT 3' | 775–797 |
(XM_875452) | Reverse | 5' ACCTTGTACTTCGAGGTGGAGC 3' | 842–821 |
TFAP2B
| Forward | 5' CGAATGCCTCAATGCGTCT 3' | 1090–1108 |
(BC120374) | Reverse | 5' CCCATTTTTCGATTTGGCTC 3' | 1150–1131 |
TFAP2C
| Forward | 5' GGTGTTCTCAGAAGAGCCAAGTC 3' | 1016–1038 |
(BC120401) | Reverse | 5' GACATAGGCAAAGTCCCGAGC 3' | 1186–1166 |
GAPDH
| Forward | 5' AAGGCCATCACCATCTTCCA 3' | 178–197 |
(U85042) | Reverse | 5' CCACTACATACTCAGCACCAGCAT 3' | 253–230 |
In situ hybridization
Results
Correlations in the microarray data
Gestation Days | EEM-COT/r value | ENDO-CAR/r value |
---|---|---|
Non-Pregnant | --- | ≥ 0.87 |
Day25 | ≥ 0.87 | ≥ 0.91 |
Day60 | ≥ 0.85 | ≥ 0.86 |
Day150 | ≥ 0.85 | ≥ 0.92 |
Day250 | ≥ 0.81 | ≥ 0.70 |
Cluster analysis of global gene expression in bovine placenta
General gene expression
Specific genes and their expression patterns in the clusters
Accession No. | Gene Name |
---|---|
Cluster 1 | |
NM_175797 | ARHGDIB: Rho GDP dissociation inhibitor beta |
NM_174506 | BCKDHA: branched chain alpha-keto acid dehydrogenase |
NM_001034046 | COX5B: Cytochrome c oxidase subunit Vb |
NM_173906 | DCN: Decorin |
NM_001040498 | JSP.1: MHC Class I JSP.1 |
NM_001015556 | RPL18: Ribosomal protein L18 |
NM_001015531 | RPS5: Ribosomal protein S5 |
NM_174464 | SPARC: secreted protein, acidic, cysteine-rich |
NM_001002885 | TMSB4X: Thymosin beta 4, X chromosome |
NM_174491 | YWHAE: 14-3-3 epsilon |
Cluster 2 | |
NM_181007 | CSH1: Placental lactogen |
AB098803 | LOC404051: Similar to thrombin inhibitor |
AB098909 | SERPINB6: Serpin peptidase inhibitor clade B member 6 |
NM_174411 | PAG1: Pregnancy-associated glycoprotein 1 |
NM_176616 | PAG5: Pregnancy-associated glycoprotein 5 |
NM_176618 | PAG7: Pregnancy-associated glycoprotein 7 |
NM_174159 | PRP1: Prolactin-related protein 1 |
M27239 | PRP2/4: Prolactin-related protein 2/4 |
NM_177488 | SULT1E1: Sulfotransferase family 1E estrogen-preferring member 1 |
NM_174623 | TMSB10: Thymosin, beta 10 |
Cluster 3 | |
NM_173979.3 | ACTB: Actin, beta |
NM_181018 | CLCA3: Chloride channel, calcium activated, family member 3 |
NM_174333 | GRP58: Glucose regulated protein 58 kD |
M83104 | LOC515773: Cytochrome b-5 reductase |
XM_870635 | FTH1: Ferritin heavy polypeptide 1 |
NM_001038163 | MGC133894: Similar to Tubulin alpha-3 chain |
NM_174600 | SLC1A3: Solute carrier family 1 |
NM_001010995 | TPM2: Tropomyosin 2 |
M62428 | UBC: Polyubiquitin |
NM_174217 | VIL2: Villin 2 |
Cluster 4 | |
NM_001012519 | AKR1B1: Aldose reductase |
NM_174800 | CFDP2: craniofacial development protein 2 |
AF013213 | EEF1A1: Eukaryotic translation elongation factor 1 alpha 1 |
NM_174550 | HSPA1A: Heat shock 70 kD protein 1 |
NM_174345 | HSPA8: Heat shock 70 kDa protein 8 |
NM_001025569 | HSPB1: Heat shock 27 kDa protein 1 |
NM_174087 | IGF2: Insulin-like growth factor 2 |
NM_174378 | LALBA: Lactalbumin, alpha |
XM_583697 | LOC507139: Similar to grancalcin |
NM_180999 | LYZ: Lysozyme |
Cluster 5 | |
NM_181003 | AQP4: Aquaporin 4 |
NM_174257 | CALB3: Calbindin 3 |
AB099882 | COL12A1: Collagen, type XII, alpha 1 |
NM_001034039 | COL1A1: Collagen, type I, alpha 1 |
NM_174770 | GPX4: Glutathione peroxidase 4 |
NM_001033610 | KRT8: Keratin 8 |
XM_588040 | LOC510833: Similar to Collagen alpha 1(III) |
NM_174459 | SEPP1: Selenoprotein P-like protein precursor |
NM_174472 | TIMP2: Tissue inhibitor of mettaloproteinase 2 |
AF241780 | TKDP5: Trophoblast Kunitz domain protein 5 |
Cluster 6 | |
NM_001046249 | CALM1: Calmodulin 1 |
NM_001038220 | CORO2A: Coronin, actin binding protein, 2A |
K00800 | FN1: Fibronectin 1 |
NM_174076 | GPX1: Glutathione peroxidase 1 |
NM_174343 | HSD3B: HSD3B protein |
NM_174556 | IGFBP3: Insulin-like growth factor binding protein 3 |
XM_614707 | LOC534812: Similar to Heat shock protein HSP 90-alpha |
NM_001033608 | MIF: Macrophage migration inhibitory factor |
NM_001015592 | PFN1: Profilin 1 |
NM_174471 | TIMP1: Tissue inhibitor of metalloproteinase 1 |
Cluster 7 | |
NM_174716 | ANXA2: Annexin A2 |
NM_001045935 | CTDSP2: CTD small phosphatase 2 |
NM_174642 | HSD11B2: Hydroxysteroid (11-beta) dehydrogenase 2 |
XM_612863 | LOC533452: Similar to Alpha-1 catenin |
NM_174798 | MSX1: Msh homeo box 1 |
NM_174115 | MUC1: Mucin 1 |
NM_205775 | TKDP4: Trophoblast Kunitz domain protein 4 |
BE477825 | VCL: Vinculin |
NM_174216 | VEGF: Vascular endothelial growth factor |
NM_174487 | VEGFB: Vascular endothelial growth factor B |
Cluster 8 | |
NM_173902 | CLU: Clusterin |
NM_174520 | COL1A2: Collagen, type I, alpha 2 |
NM_174029 | CST3: Cystatin C |
NM_174031 | CTSB: Cathepsin B |
NM_176612 | HMGB1: High-mobility group box 1 |
NM_174092 | IL1A: Interleukin 1, alpha |
XM_598260 | LAMB1: Laminin, beta 1 |
NM_174707 | MGP: Matrix Gla protein |
NM_174030 | CTGF: Connective tissue growth factor |
NM_174797 | UMP: Uterine milk protein |
Cluster 9 | |
NM_175784 | ANXA1: Annexin I |
AF515786 | B4GALT1: Glycoprotein-4-beta-galactosyltransferase 2 |
NM_174032 | CTSL: Cathepsin L |
NM_176608 | GHR: Growth hormone receptor |
NM_178319 | GRP: Gastrin-releasing peptide |
NM_174125 | NPPC: Natriuretic peptide precursor C |
NM_176621 | PAG10: Pregnancy-associated glycoprotein 10 |
NM_176619 | PAG8: Pregnancy-associated glycoprotein 8 |
NM_174182 | SELL: Selectin L |
NM_176669 | STC1: Stanniocalcin 1 |
Cluster 10 | |
BC109863 | APOD: Apolipoprotein D |
NM_176648 | CAPZB: Capping protein (actin filament) muscle Z-line, beta |
NM_176644 | CYP11A1: Cytochrome P450, family 11, subfamily A, polypeptide 1 |
NM_174363 | INHBA: Inhibin, beta A |
NM_174100 | LDHB: Lactate dehydrogenase B |
NM_001034053 | LMNA: Lamin A |
XM_615304 | LOC541253: Similar to Limbic system-associated membrane protein |
NM_174745 | MMP2: Matrix metalloproteinase 2 |
NM_174409 | OSTF1: Osteoclast stimulating factor 1 |
BC102667 | S100A11: S100 calcium binding protein A11 |