Total RNA was extracted according to the manufacturers' instructions using RNeasy Plus kit (Qiagen, Hilden, MD, USA) for fibroblasts or Trizol (Invitrogen, Thermo Scientific) for monocytes. Random hexamers and the M-MuLV reverse transcriptase enzyme (Thermo Scientific) were used and RT-QPCR was performed in triplicate using Maxima SYBR Green/Rox (Thermo Scientific) and analysed on the Mx3005P QPCR system (Agilent Technologies). The relative mRNA expression was normalized to ACTB, HPRT and GAPDH and calculated using the comparative Ct method54 (link). For primers see Supplementary Table 5.
Allaoui R., Bergenfelz C., Mohlin S., Hagerling C., Salari K., Werb Z., Anderson R.L., Ethier S.P., Jirström K., Påhlman S., Bexell D., Tahin B., Johansson M.E., Larsson C, & Leandersson K. (2016). Cancer-associated fibroblast-secreted CXCL16 attracts monocytes to promote stroma activation in triple-negative breast cancers. Nature Communications, 7, 13050.
Other organizations :
Lund University, UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, Peter MacCallum Cancer Centre, University of Melbourne, MUSC Hollings Cancer Center, Medical University of South Carolina, Skåne University Hospital
No independent variables are explicitly mentioned.
dependent variables
Relative mRNA expression of target genes
control variables
Extraction of total RNA using RNeasy Plus kit (Qiagen) for fibroblasts or Trizol (Invitrogen, Thermo Scientific) for monocytes
Use of random hexamers and M-MuLV reverse transcriptase enzyme (Thermo Scientific) for reverse transcription
RT-QPCR performed in triplicate using Maxima SYBR Green/Rox (Thermo Scientific) and analyzed on the Mx3005P QPCR system (Agilent Technologies)
Normalization of relative mRNA expression to reference genes ACTB, HPRT, and GAPDH
positive controls
No positive controls are explicitly mentioned.
negative controls
No negative controls are explicitly mentioned.
Annotations
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