Reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) analysis was performed as described previously64 (link). Reactions were performed in triplo for determination of the levels of Yes, Lyn, Frk, Lck, Fgr, Hck, Blk and CyclofilinA (CF; control) in A431 cells. Cells were grown in complete medium and lysed in RNA-Bee (Tel-test Inc.) or Trizol reagent (Ambion #15596018), following manufacturer’s instructions. Total RNA was separated from DNA and proteins by the addition of chloroform (Sigma-Aldrich) and subsequent centrifugation at 12,000 × g for 15 min at 4 °C. The RNA was precipitated with isopropanol and washed with 70% ethanol. Integrity of the isolated RNA was assessed by agarose gel electrophoresis.
First strand cDNA synthesis was performed with 3 μg of total RNA using the first strand cDNA synthesis kit K1612 (Thermo Fisher Scientific) following manufacturer’s directions. The PCR reactions were run using SYBR Advantage qPCR premix (Clontech) on a 7500 Fast Real-Time PCR system (Applied Biosystems) and the primers (IDT) which were used, are shown in TableS2 . Relative mRNA quantities were obtained using the 2−ΔCt method. The ΔCt is the obtained Ct value of the SFK minus the obtained Ct value of CF (endogenous control).
First strand cDNA synthesis was performed with 3 μg of total RNA using the first strand cDNA synthesis kit K1612 (Thermo Fisher Scientific) following manufacturer’s directions. The PCR reactions were run using SYBR Advantage qPCR premix (Clontech) on a 7500 Fast Real-Time PCR system (Applied Biosystems) and the primers (IDT) which were used, are shown in Table
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