For reverse transcription–polymerase chain reaction (RT-PCR),
total RNA was extracted from PE and NPE cells with a kit (RNeasy;
Qiagen, Chatsworth, CA) including DNase I digestion, according to the
instructions of the manufacturer. First-strand cDNA was produced by
M-MLV reverse transcriptase (Promega, Madison, WI) using 1 μg of RNA
and 0.2 μg oligo (dT)15. Each RNA preparation
was controlled for the presence of genomic DNA and subjected to
digestion (RNase ONE; Promega) to show specific amplification from
cDNA. PCR was performed with cDNA corresponding to 50 ng total RNA, 200μ
M dNTP, 1 μM of each primer, 1.5 mM MgCl2,
and 2 U Taq-DNA-polymerase (Promega) in a total volume of 40μ
l with 40 cycles of 94°C (45 seconds), 59°C (50 seconds), and
72°C (45 seconds). Primer sequences were 5′-ACCATGTGCACCCTCATCAC-3′
for both human and rat slc-1 sense primers and
5′-TCTCACAGAGCACGATGTAC-3′ and 5′-TCTCACACAGAGCACTATGTAC-3′ for human
and rat slc-1 antisense primers, respectively. Because the slc-1 sequence is very highly conserved between species, the
rat slc-1 primers could be used for detection of slc-1 transcripts in porcine PE cells. PCR products were
resolved on a 1.5% agarose gel and visualized by ethidium bromide
staining.