Figure 3.

Analyzes of the mRNA expression of the short and long isoform in human cells

A) PCR amplification of ”short” and ‘long’ LARP1 isoforms using specific primers hybridizing to exon 1 of the respective isoform. S1/S2 and L1/L2 stand for primer pairs detecting SI- or LI-LARP1, respectively. T stands for total RNA detected using a primer pair annealing to constitutively expressed Exon 7. HK stands for housekeeping gene OAZ1. B) PCR product containing the complete open reading frame of long LARP1 was generated using gene-specific primers annealing to 5ʹ- and 3ʹ- end of the open reading frame. Amplification products were purified and sequenced. Junction of ‘long’ LARP1 specific exon 1 and common exon 2 is given in chromatograms. C) Systematic analysis of mRNA expression levels of short and long LARP1 isoforms in NCI-60 cancer panel using quantitative PCR. Specific primers for short and long LARP1 were used. Results of one representative primer pair for each isoform are shown. Dark and light-coloured bars represent proportion of SI- or LI-LARP1 expression, respectively. D) Expression of LI-LARP1 and absence of SI-LARP1 mRNA was validated in SK-OV-3, OVCAR-8, MCF-7, and 501-MEL cells using quantitative PCR. Resulting data were normalized against OAZ1. (E) Quantitative PCR analysis of expression of short LARP1 using random or oligo-(dT) primers for reverse transcription suggest that mRNA is missing poly-(A) tail. All qPCR data were normalized against OAZ1 and relative expression values were calculated using 2-Δct method.