Hs gapdh 2 sg quantitect primer assay

Total RNA was isolated from cells using the RNeasy^® Plus Mini Kit (Qiagen, Foster City, CA, USA) according to the manufacturer's protocol. For 2D cell cultures, 2x10⁵ to 1x10⁶ cells were lysed with RLT buffer according to the manufacturer's protocol. For 3D culture, 4x10⁵ cells were seeded in 12-well plates on matrigel, as described for protein extraction. Pelleted spheroids were lysed in 350 μl RLT buffer according to the manufacturer's protocol. RNA samples were quantified using a Nanophotometer (Implen, Munich, Germany) at OD 260/280 nm. Q-PCR was performed with QuantiTect Primer Assays^® for SYBR^® Green-based expression analysis (Qiagen) using a Cfx96 device (Biorad) according to the manufacture's protocol for one-step RT-PCR. Primers used were Hs_NRG1_1_SG QuantiTect Primer Assay, Hs_NRG2_1_SG QuantiTect Primer Assay and Hs_GAPDH_2_SG QuantiTect Primer Assay (all Qiagen). Changes in the relative expression level were calculated using the 2-ΔΔCt method (Biorad CFX manager software 3.1.). GAPDH was used as the endogenous control gene for normalization.

MDA-MB-231 cells were infected with oncolytic adenoviruses at MOI-100, and RNA was isolated 32 h post infection as above. cDNA was prepared using the Cloned AMV First-Strand Synthesis Kit (Invitrogen) or FireScript RT cDNA Synthesis Kit (Solis BioDyne) according to the manufacturer’s recommendations. qRT-PCR was performed on a Roche LightCycler 480 system using 5× HOT FIREPol EvaGreen qPCR Mix Plus (no ROX). Human FOXP1, MET, and PTGS2 primers were purchased as custom oligonucleotides from Invitrogen (Table S3). Hs_GAPDH_2_SG QuantiTect Primer Assay (QIAGEN, Hilden, Germany) was used for GAPDH. All reactions were done in duplicate. GAPDH-normalized FOXP1, MET, or PTGS2 gene expression was calculated using the ΔCt method; knockdown compared to irrelevant control virus-infected cells was calculated using the ΔΔCt method.