Icycler iqtm software

MDA-231 and MDA-1833 cells were seeded in T75 flasks (Sarsted) and cultured as described above. When 80% confluence was reached, cells were washed with PBS and lysed with Trizol (Invitrogen, Cat#15596-018) and kept on ice. Total RNA from six independent replicates was extracted using the Direct-zol™ RNA miniPrep, according to the manufacturer’s protocol (Zymo Research, Tustin, CA, USA, Cat#ZY-R2052). RNA final concentration and purity (OD_260/280) was determined using a NanoDrop 2000 instrument (NanoDrop Technologies, Wilmington, NC, USA). RNA was reverse transcribed into cDNA using the NZY First-Strand cDNA Synthesis Kit (NZYTech, Cat#MB12501, Lisbon, Portugal), according to the manufacturer’s protocol. qRT-PCR experiments were run using an iCycler iQ5 PCR thermal cycler (Bio-Rad Laboratories, Lisbon, Portugal) and analyzed with the iCycler IQ^TM software (Bio-Rad). A personalized PrimePCR array (Bio-Rad Laboratories, Lisbon, Portugal) was designed to analyze the range of adrenergic receptors. Target gene expression was quantified using the cycle threshold (Ct) values and relative mRNA expression levels were calculated as follows: 2^(Ct reference gene − Ct target gene). Human β2-microglobulin (β2M) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as reference genes. Both target and reference genes were amplified with efficiencies between 100 ± 5%.

Total RNA was extracted using the Direct-zol™ RNA miniPrep according to the manufacturer’s protocol (Zymo Research). RNA final concentration and purity (OD260/280) was determined using a NanoDrop 2000 instrument (NanoDrop Technologies). RNA was reverse transcribed into cDNA using the NZY First-Strand cDNA Synthesis Kit (NZYTech), according to the manufacturer’s protocol. For the analysis of neurotrophins expression levels, a personalized PrimePCR array (Bio-Rad Laboratories) was performed. qRT-PCR experiments were run using an iCycler iQ5 PCR thermal cycler (Bio-Rad Laboratories) and analyzed with the iCycler IQTM software (Bio-Rad). Target gene expression was quantified using the cycle threshold (Ct) values and relative mRNA expression levels were calculated as follows: 2^(Ct reference gene − Ct target gene). GAPDH was used as a reference gene. Both target and reference genes were amplified with efficiencies between 100% ± 5%.

Total RNA was extracted using the Direct-zol™ RNA miniPrep according to the manufacturer’s protocol (Zymo Research). RNA final concentration and purity (OD260/280) was determined using a NanoDrop 2000 instrument (NanoDrop Technologies). RNA was reverse transcribed into cDNA using the NZY First-Strand cDNA Synthesis Kit (NZYTech), according to the manufacturer’s protocol. For the analysis of repulsive cues, a personalized PrimePCR array (Bio-Rad Laboratories) was performed. qRT-PCR experiments were run using an iCycler iQ5 PCR thermal cycler (Bio-Rad Laboratories) and analyzed with the iCycler IQTM software (Bio-Rad). Target gene expression was quantified using the cycle threshold (Ct) values and relative mRNA expression levels were calculated as follows: 2^(Ct reference gene − Ct target gene). Human β-2-microglobulin was used as a reference gene. Both target and reference genes were amplified with efficiencies between 100% ± 5%.