Background
Results
Differentially expressed genes
RT-qPCR
Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways
IPA canonical pathway, tox list, and upstream regulators
Discussion
Inhibited luteinization of cocultured GCs under elevated NEFAs levels
Apoptosis and Inflammation
Impaired mitochondrial function
GCs protect COCs against exposure to excessive NEFA levels
Effects of the high levels of NEFAs on different ovarian cell types
Conclusions
Materials and methods
Chemicals
Preparation of NEFAs
Collection of GCs and COCs
Preparation of granulosa cell monolayers
Co-culture of COCs and granulosa cell monolayers
RNA extraction
Manipulation of RNA microarrays
Data analysis
Quantitative Real-Time RT-qPCR
Target gene | Accession number | Forward primer (5’-3’) | Reverse primer (5’-3’) | Product size | Annealing Temperature |
---|---|---|---|---|---|
AGPS
| XM_021075128.1 | GACGCAGACTTATGATGCAGG | AGCCACTGCTTCCGTAACT | 180 bp | 57 °C |
MAP3K8
| XM_021064737.1 | ATGGAACCGTGGAGGACTTG | CGGAGCCGATATTCCTGTAAGT | 192 bp | 57 °C |
TLR6
| NM_213760.2 | CTCTCATGGCACAGCGAACT | CACATCATCCTCTTCAGCGACT | 125 bp | 59 °C |
AGO1
| XM_005665209.3 | ACGCTGTTACCTCACTGGATAG | GGCCAGATGTGACAAGATGAGG | 244 bp | 59 °C |
IL33
| NM_001285978.1 | ATGGTGGTGGTGATCATCGGA | ACACTCCAGGATGGCTCTTACA | 201 bp | 59 °C |
GAPDH
| NM_001206359.1 | ACTGGTGTCTTCACGACCAT | GGAGGCATTGCTGACGATCT | 159 bp | 57 °C |
STAR
| NM_213755.2 | CCCTTGGACAGGAGCTGAAC | GGCCAGATCTTGGTCACTGT | 134 bp | 57 °C |
CYP11A1
| NM_214227.1 | AGACACTGAGACTCCACCCCA | GACGGCCACTTGTACCAATGT | 110 bp | 57 °C |
HSD3B1
| NM_001004049.2 | GATCGTCCACTTGTTGCTGG | GATCTTGCTCTGGAGCTTAGAA | 109 bp | 57 °C |
CYP19A3
| NM_214431.1 | GTGGACGTGTTGACCCTCAT | TATGCACGAGGGCACTTTCG | 94 bp | 57 °C |
SDHA [61] | XM_021076930.1 | CACACGCTTTCCTATGTCGATG | TGGCACAGTCAGCTTCATTC | 94 bp | 57 °C |
PPIA [61] | NM_214353.1 | AAAACTTCCGTGCTCTGAGC | TTATGGCGTGTGAAGTCACC | 112 bp | 59 °C |