Quantitaive RT-PCR (qRT-PCR)

Total RNA was extracts with an RNeasy Mini Kit (Qiagen, Hiden, DE), according to the manufacturer’s instructions. qRT-PCR was performed on MMP2. GAPDH and actin were used as internal controls. All reactions were performed by using the i-Script One Step RT-PCR Sybr Green Kit (BioRad) according to the manufacturer’s instructions. The real time RT-PCR primers are summarized in Table 1. qRT-PCR for MMP2 was performed in a total of 25 µl from a mixture containing 0.5 µl of Taq Polymerase with Sybr green mix, 12.5 µl of 2X Buffer, 4.5 µl of forward primer (900 nM), 4.5 µl of reverse primer (900 nM), 1000 ng of RNA for MMP2 gene. Reactions were run in BioRad CFX-96 detector system under the following conditions: 40 cycles of 95 °C for 10 s, 60 °C for 30 s, after pre-incubation at 95 °C for 5 s. All reactions were performed in triplicate. The specificity of the amplification reactions was confirmed by melting-curve analysis. The fold changes in the mRNA expression level of MMP2 in the two cell lines and control sample were compared using the DDCq method (BioRad CFX Manager Software).

Real time RT-PCR for in vivo mabs injection was performed as described.⁴⁰ Briefly, total RNA was extracted by liquid nitrogen homogenization with TRIzol reagent (Life Technologies) following the manual instruction. The obtained RNA was cleaned and DNase treated with RNeasy Mini Kit (Quiagen). Single-stranded cDNA was synthesized with SuperScript First-Strand RT-PCR system (Life Technologies), according to the protocol supplied by the manufacturer. Real time PCR was performed using SYBR GREEN PCR Master Mix (PE Applied Biosystems Foster City, CA, USA) according to the supplied method by intron-spanning primers: