Quantitative PCR Analysis of GLI1 Isoforms

We extracted total RNAs from cultured cells or tumorous tissues using Tri-RNA extraction reagent (Sigma, St. Luis, MO, USA), and performed real-time quantitative PCR analyses according to a previously published procedure [57 (link)]. For each sample, we used triplicates and used the comparative C_T (ΔΔCT) as described by the manufacturer (Applied Biosystems/ Fisher Scientific). The relative amount of target (2^−ΔΔCT) was obtained by normalizing to an endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and relative to a calibrator. All primers and probes were purchased from Applied Biosystems/Fisher Scientific). RT-PCR was used to detect possible existence of GLI1 isoforms in the pancreatic cancer cell lines. Primers 5′-TGTTCAACTCGATGACCC-3′ and 5′-GTCATGGGGACCACAAGG-3′ were used to detect wild type GLI1 (500 bp) and truncated GLI1 (tGLI1) (377 bp). Primers 5′-GGCATCCGACAGAGGTGAGATGGAC-3′ and 5′-GAGCCCAGCGCCCAGACAGA-3′ or 5′-CTGTCTCAGGGAACCGTGGGTCTTTGT-3′ were used to detect full-length GLI1 or GLI1ΔN in pancreatic cancer cell lines.