RNA extraction, reverse transcription, and real-time PCR

Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNAs were amplified and quantified using an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with SYBR Green I dye (Molecular Probes, Eugene, OR, USA). Expression data were normalized to the geometric mean of the housekeeping gene β-actin. The primers were selected as follows:

Sam68, forward 5′-ATGAAGCTTATGGCCAGGAC-3′ and reverse 5′-CAGAAGCCAGAATGCAGAGT-3′, β-actin, forward 5′-TGGCACCCAGCACAATGAA-3′ and reverse 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′.

Real-time PCR was performed according to standard methods, as described previously [13].