Total RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) for ALCAM analysis
The cDNA templates were then diluted 1:5 with sterile water and amplified by PCR using Taq polymerase (Takara Bio, Shiga, Japan), and specific primers based on mRNA sequences of porcine ALCAM (GenBank accession number AJ311681; forward, 5'-CCT TCA GGT CCT CCA CAA AG-3'; reverse, 5'-ATG TGA TGT TGC CAT CTG GA-3') and porcine ribosomal protein L7 (RPL7), (GenBank accession number NM 001113217; forward, 5' AAG CCA AGC ACT ATC ACA AGG AAT ACA-3'; reverse, 5'-TGC AAC ACC TTT CTG ACC TTT GG-3') were designed to amplify cDNA of 302 bp and 172 bp, respectively.
To analyze levels of ALCAM mRNA in the uterine endometrium, real-time RT-PCR was performed using the Applied Biosystems StepOnePlus System (Applied Biosystems, Foster City, CA) and the SYBR Green method.
Sense and antisense ALCAM riboprobes labeled with DIG-UTP were denatured for 5 min at 80[degrees]C and added to the hybridization mix.
Data from real-time RT-PCR for ALCAM expression were subjected to least squares ANOVA using the General Linear Models procedures of SAS (Cary, NC).
Expression and regulation of ALCAM mRNA in the uterine endometrium during the estrous cycle and pregnancy in pigs
To determine ALCAM mRNA levels in the uterine endometrium during the estrous cycle and pregnancy in pigs, we conducted real-time RT-PCR analysis.
Localization of ALCAM mRNA in the uterine endometrium during the estrous cycle and pregnancy
To determine which cell types express ALCAM mRNA in the uterine endometrium during the estrous cycle and pregnancy and in conceptuses, we performed in situ hybridization analysis using uterine endometrial tissues from D12 and D15 of the estrous cycle and pregnancy and conceptuses from D12 of pregnancy.
Expression of ALCAM in the conceptus during early pregnancy