Quantification of alternatively and correctly spliced 5-LO mRNA.

<p>Time courses of 5-LO mRNA induction in MM6 cells cultured with (diff) and without (undiff) TGFβ (1 ng/ml) and calcitriol (50 nM). Total RNA was extracted and cDNA was prepared using random hexamer primers for the PCR analysis of correctly spliced 5-LO pre-mRNA applying primer pair C (exon 2 to exon 5) and E (exon 10 to exon 14). 5-LOΔ3 mRNA was determined by primer pair G. For the specific analysis of the mature 5-LO transcripts, the samples were reverse transcribed with oligo-dT priming and the cDNA was amplified with primer pair F (exon 13 to 3′UTR). β-actin mRNA served as constitutively expressed control. The relative changes to day 0 are given as the mean+SE of three independent experiments. Time courses of 5-LO mRNA in (A) differentiated and (B) undifferentiated MM6 cells. (C) Time courses of 5-LOΔ3 mRNA in MM6 cells cultured with (diff) and without (undiff) TGFβ (1 ng/ml) and calcitriol (50 nM). 5-LOΔ3 cDNA was analyzed using primer pair G. For the analysis of correctly spliced 5-LO mRNA the primer pair C was used.