Total RNA extraction and cloning of porcine ABCC1, ABCC9, SLCO4C1, and SLCO5A1 cDNAs
The cDNA templates were then diluted 1:4 with nuclease-free water and amplified by polymerase chain reaction (PCR) using Taq polymerase (Takara Bio, Shiga, Japan), and specific primers based on porcine ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNA sequences.
The levels of expression of ABCC1, ABCC9, SLCO4C1, and SLCO5A1 genes in endometrial and chorioallantoic tissues were analyzed by real-time RT-PCR using the Applied Biosystems StepOnePlus System (Applied Biosystems, Foster City, CA, USA) and SYBR Green method.
Non-radioactive in situ hybridization was performed to deter mine the localization of ABCC1 and ABCC9 expression in the uterine endometrium as previously described with some modifications .
Explant tissues were then collected and total RNA or protein was extracted for real-time RT-PCR and immunoblot analysis, respectively, to determine the expression levels of ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNAs.
Data from real-time RT-PCR for ABCC1, ABCC9, SLCO4C1, and SLCO5A1 expression during the estrous cycle and pregnancy were subjected to least squares analysis of variance using the general linear models procedures of SAS (Cary, NC, USA).
On D12 and D15 post-estrus, expression of ABCC1 mRNA was affected by pregnancy status (p<0.
Expression of ABCC1, ABCC9, SLCO4C1, and SLCO5A1 mRNAs in conceptuses during early pregnancy and in chorioallantoic tissues during late pregnancy
As shown in Figure 2B, levels of ABCC1 and ABCC9, but not SLCO4C1 and SLCO5A1 mRNAs changed in chorioallantoic tissues on D30, D60, D90, and D114 of pregnancy (quadratic effect of day for ABCC1, p = 0.