RNA isolation was carried out by using guanidine isothiocyanate (TRIzol reagent, Invitrogen Corporation, Carlsbad, CA, USA). Cells were lysed in 1 mL of TRIzol reagent and then 200 µL of chloroform was added. The mixture was vigorously shaken, incubated at room temperature for 10–15 min and centrifuged at 12,000× g for 15 min. The aqueous phase was collected, and the RNA was precipitated by isopropanol addition. The pellet, previously washed in 75% ethanol, was dissolved in 10 mM Tris-HCl pH 7.5, 1 mM EDTA. Purified RNA samples were processed with the DNA-free DNA Removal Kit (Invitrogen, Thermo Fisher Scientific, Milan, Italy) to remove contaminating DNA. RNAs (2 µg) were reverse transcribed (RT) with or without the ImProm-II™ Reverse Transcriptase (Promega Italia, Milan, Italy) using oligo-dT primers in a standard 20 µL reaction. Then, 1 µL of this reaction mixture was used as a template in gene-specific amplifications performed on a StepOnePlus Real-Time PCR System using the StepOne software v2.3 (Thermo Fisher Scientific, Milan, Italy). The sequence of primers used in the amplification of FOXN3, CDKN1A and ACTB mRNAs have been previously described [27 (link)]. The sequence of primers used in the amplification of ABCB1, ABCC1, and ABCG2 mRNAs have been previously described [54 (link)]. PCR amplifications were performed in a 50 µL volume containing 25 µL SYBR green PCR master mix (Thermo Fisher Scientific, Milan, Italy) containing the ROX internal passive reference dye, 0.5 µM of each primer, and optimized MgCl2 concentration between 1.5 and 3 mM. All determinations were performed in triplicate wells. Samples in which the RNA was not reverse-transcribed gave CT values comparable to those obtained in the no-template control well. Endpoint amplified products were subjected to melt-curve analysis. The relative quantity of the target transcript in the sample was calculated with respect to the reference ACTB mRNA using a comparative CT (ΔΔCT) method. The relative value was expressed as 2−ΔΔCT.
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