Quantitative real-time PCR analysis

qRT-PCR was performed on Bio-Rad IQ5 instrument (Bio-Rad, Madrid, Spain) and MxPro Mx3005p qPCR thermal cycler (Stratagene, Madrid, Spain) using, respectively, SYBR Green (Bio-Rad, Madrid, Spain) and Dynamo SYBR Green (Finnzymes, Finland) master mix as described previously [44 (link), 45 (link)]. The primer pairs were located in different exons to rule out genomic DNA amplification. Each primer pair used yielded a single peak of dissociation on the melting curve and a single band with the expected size on PAGE gels. Identity of the PCR products was confirmed by sequencing. NT and non-RT RNA template reactions were used as negative controls. All PCR setups were performed at least in triplicate. Relative quantifications were calculated with the comparative ΔCt cycle method with normalization to the expression of housekeeping genes coding for ribosomal protein L19 (Rpl19), β-actin, glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and β-D-glucuronidase (Gusb). The efficiency of target and reference amplifications was tested to be approximately equal. miRNA qRT-PCR was performed using Exiqon LNA microRNA qRT-PCR primers and detection kit (Exiqon, Madrid, Spain) according to manufacturer's guidelines. All reactions were run in triplicate using 5S as normalizing control. Primer sequences and additional data are available upon request.