Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

Messenger RNA expression of the major RASSF1 variants RASSF1A, RASSF1B and RASSF1C was determined in 14 xenografted PDAC and 8 PDAC cell lines. RNA samples were retrotranscribed to cDNA using the First Strand cDNA Synthesis Kit (Roche). A reverse transcriptase minus cDNA was prepared for each sample as a control. QRT-PCR was carried out as previously described [36] in 25 μl total volume containing 4 ng of cDNA, 1x Power SYBR Green I Master Mix (Applied Biosystems), 400 nM of each primer. After a starting denaturation for 10 min at 95 °C, 45 PCR cycles (15 s 95 °C and 1 min 60 °C) have been performed on ABI PRISM 7900HT SDS instrument (Applied Biosystems). The relative expression level was calculated using transcript level of RPLPO as reference gene and the standard (=1) was the average of the levels of expression of all samples. QRT-PCR data analysis was performed according to the comparative method following the User Bulletin #2 (Applied Biosystems).