Omnizol

We extracted total RNA from cell cultures using Omnizol (EuroClone, Italy) according to the manufacturer’s protocol; we then measured mRNA levels by RT-PCR amplification.
We used sequences of mRNAs from the Nucleotide Data Bank (National Center for Biotechnology Information, MA, USA) to design primer pairs for real-time RT-PCR reactions (Primer Express, Applied Biosystems, Italy); primer sequences are available upon request. We used appropriate regions of HPRT and/or GAPDH cDNA as controls and ran real-time PCR assays on an Opticon 4 machine (MJ Research, MA, USA). We carried out reactions according to the manufacturer’s instruction using an SYBR green PCR master mix and used the 2^-ΔΔCT method as a relative quantification strategy for quantitative real-time PCR data analysis.

We extracted total RNA from cell cultures using Omnizol (EuroClone, Italy), according to the manufacturer’s protocol, and measured mRNA levels by RT-PCR amplification.
We used sequences of mRNAs from the Nucleotide Data Bank (National Center for Biotechnology Information, MD, USA) to design primer pairs for real-time RT-PCR reactions (Primer Express, Applied Biosystems, Italy); primer sequences are available upon request. We used appropriate regions of HPRT and/or GAPDH cDNA as controls and ran real-time PCR assays on an Opticon 4 machine (MJ Research, MA, USA). We carried out reactions according to the manufacturer’s instructions using a SYBR green PCR master mix and used the 2^-ΔΔCT method as a relative quantification strategy for quantitative real-time PCR data analysis.

RNA was extracted from cell samples with Omnizol (EuroClone, Pero (MI), Italy). The mRNA levels were determined by RT-PCR and by 5X All-In-One RT MasterMix (ABM, Richmond, BC, Canada), and the real-time PCR assays were carried out with BrightGreen 2X qPCR MasterMix (ABM, Vancouver, BC, Canada) and run on a LineGene 9600 PCR detection system (Bioer Technology, Hangzhou, China). All of the reagents were used according to the manufacturer’s instructions. We designed primer pairs for RT-PCR reactions with Primer Express software (Applied Biosystems, Milan, Italy) and used the mRNA sequences as templates from the Nucleotide DataBank (National Center for Biotechnology Information, Bethesda, MD, USA) to design primer pairs. Primer sequences are available upon request. We used the 2-ΔΔCT method as a relative quantification strategy for quantitative real-time PCR data analysis.