Quantitative RT-PCR Assay Validation
The presence of skunk LIF and cyclophilin in total uterine RNA extracts was demonstrated by RT-PCR. Specific primers yielded PCR products of the expected size: 398 bp for LIF and 195 bp for cyclophilin. Nucleotide sequence of the amplified region of skunk LIF mRNA was 86.4% identical to that of human LIF mRNA, and 87.3% similar in protein sequence. The nucleotide sequence of the cloned portion of skunk cyclophilin was 78.9% similar to that of human cyclophilin. No signal was detected in samples that lacked reverse transcriptase, whereas specific binding of LIF and cyclophilin DNA probes was detected in Southern blots of amplified samples containing reverse transcriptase (data not shown). buy cipro
Parallelism between the plasmid standards for LIF and cyclophilin and uterine cDNA dilution curves was demonstrated (data not shown). These results verified that LIF and cyclophilin each amplified from the plasmid standards and cDNA samples with comparable efficiency. Sensitivity of the RT-PCR assay was below 10 ag for cyclophilin and 1 fg for LIF per 10 ng cDNA. Intraassay variation of the entire procedure (reverse transcription and PCR) was 34.8 ± 15.6%. The average intraassay coefficient of variation between duplicate cDNA samples in the PCR procedure was 8.3 ± 9.2% for the pregnancy series and 8.2 ± 5.8% for the hormone treatments.