Identification and Characterization of a Novel Nontranslated Sequence Variant of the Human Intestinal Di-/Tripeptide Transporter, hPEPT1
Figure 2
hPEPT1-RFI mRNA is expressed in Caco-2 cells and human tissues. (a) The deletion 848delT in the hPEPT1-RFI sequence was used to differentiate between the hPEPT1-RFI and the hPEPT1-RF transcripts. Absence of the T indicates that the RNA transcript is the hPEPT1-RFI version. RT-PCR was performed on human jejunum, ileum, colon, and Caco-2 mRNA (Lane 1–5), and amplicons were digested by the restriction endonuclease MnlI and analysed by agarose gel electrophoresis. The arrows indicate a 34 bp and a 70 bp segment in concordance with the hPEPT1-RFI sequence. M denotes the molecular weight DNA ladder. (b) Real-time PCR was performed with primers covering parts of hPEPT1-RFI, hPEPT1, and β-ACTIN as described in methods. RNA was sampled from Caco-2 cells grown for 7 and 24 days. Reverse transcription was performed on 0.5 μg RNA, and results are presented as means of normalized target values relative to a calibrator sample ± SD of three individual experiments, each performed in triplicates. (c) Real-time PCR was performed on human intestinal cDNA from two healthy individuals. The sample RNA was taken from the jejunal part of the intestine. Results are presented as means of normalized target values relative to the calibrator sample ± SD of each determination in triplicate.
