Human tumor metastasis pcr array

The change in the expression of genes involved in the process of neoplastic metastasis was assessed using the Human Tumor Metastasis PCR Array (Qiagen, Hilden, Germany). For each sample, Total RNA (3μg) was reverse transcribed to cDNA using the RT2 First Strand Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. Human Tumor Metastasis PCR Array was performed according to the manufacturer’s recommendations using RT2 SYBR^® Green qPCR Mastermix (Qiagen, Hilden, Germany). Recommended temperature profile used for all reactions: HotStart DNA Taq Polymerase for 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 1 min at 60 °C, followed by 10 min at 72 °C (Mx 3000p Stratagene, San Diego, CA, USA). The ΔCq value was converted as the difference between the Cq (quantitation cycle) value of the genes of interest and the Cq value of the reference housekeeping genes. Then, the ΔΔCq value was determined for each sample, and finally, the normalized expression level of the target gene was calculated from the formula: 2 − ΔΔCq. qRT-PCR-based microarray assay had as endogenous controls a list of genes: ACTB (Actin, beta), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), HPRT1 (Hypoxanthine phosphoribosyltransferase 1) and RPLP0 (Ribosomal protein, large, P0).