Rt preamp primer mix for human and mouse pcr array

To assess heterogeneity in macrophage polarization and confirm expected functional phenotypes, expression levels of genes specific to broad macrophage classes (i.e., M1-like, M2like, mixed phenotypes) were analyzed by qPCR. For 2D cultures, separate cultures of M0, M(IFN-γ), M(IL4/IL13) RAW 264.7 macrophages were maintained in 6-well plates prior to mRNA extraction. For 3D cultures, RAW264.7 macrophages or THP-1 monocytes were cultured alone or with breast cancer cells in the 3D Stacks microfluidic platform. For all experiments, mRNA was extracted at 24, 48 and 72 hours using a Dynabeads™ mRNA DIRECT™ Purification Kit (Thermo Fisher, 61012). Purified mRNA was quantified using a Qubit fluorometer (Thermo Fisher) and a Qubit™ RNA BR Assay Kit (Thermo Fisher, Q10210). mRNA was reverse transcribed using RT² PreAMP cDNA synthesis kit (Qiagen, 330451). The prepared cDNA was preamplified using the RT² PreAMP Primer Mix for Human and Mouse PCR Array (Qiagen, PBH-181Z). cDNA was analyzed by RT-qPCR using a Qiagen RT2 profiler custom panel (Qiagen, PAHS-181Z, CLAM3313, CLAH36077) following supplier instructions. Gene lists for mouse and human experiments are reported in Supplementary Table 2.