Fig 2. Confirmation of alternative NDST1 transcripts by RT-PCR and assessment of NDST1 promoter (PA region)—luciferase construct activity.
(A) Reverse transcription PCR for alternative NDST1 transcripts. Complementary DNA reversely transcribed from total extracted RNA of normal human fibroblast was used in the PCR to identify expression levels of each of the possible NDST1 transcripts. Primers flanked the sequence of 5’ UTR (exon 1) and coding region (exon 2) were expected to produce 637, 700, 688 bp PCR fragments for NDST1 mRNA transcribed from PA, PB and PC respectively. PCRs with these primers amplified only NDST1 mRNA transcribed from PA region. 18S rRNA was used as an internal control. (B) Activity of NDST1 promoter—renilla luciferase reporter construct. Renilla luciferase expression vector containing either: i) two random DNA sequences, RO1 and RO2 (negative controls); ii) NDST1 promoter (PA region); or iii) GAPDH promoter (positive control) were transiently transfected into HeLa cells. DNA fragments activity is shown as a normalized value of luminescence (produced by renilla luciferase) to fluorescence (produced by Hex). Experiments were conducted in triplicates. Ren—renilla luciferase activity, l.u.—luminescence units, f.u.—fluorescence units.