After treatment, the total RNA was isolated from U937-derived
macrophages using an RNA isolation kit (ZymoResearch, Irvine, CA) according to
the manufacturer’s instructions. Synthesis of cDNA was performed as
previously described.26 (link) The
expression of the housekeeping gene β-actin and
differentially expressed genes was analyzed via real-time PCR with a LightCycler
Instrument (LC 480, Roche Diagnostics, Mannheim, Germany), using the SYBR Green
PCR kit (Applied Biosystems). All PCR assays were performed in triplicate, and
results were expressed as fold induction relative to
β-actin. The intra-assay variability was <7%.
Data were analyzed with the LightCycler analysis software. No significant
cytotoxicity was observed using the trypan blue exclusion test of cell viability
for the macrophage cells when treated with any of the ash extracts at the level
of 1 μL extract to 1 mL medium (Figure S1 ).
macrophages using an RNA isolation kit (ZymoResearch, Irvine, CA) according to
the manufacturer’s instructions. Synthesis of cDNA was performed as
previously described.26 (link) The
expression of the housekeeping gene β-actin and
differentially expressed genes was analyzed via real-time PCR with a LightCycler
Instrument (LC 480, Roche Diagnostics, Mannheim, Germany), using the SYBR Green
PCR kit (Applied Biosystems). All PCR assays were performed in triplicate, and
results were expressed as fold induction relative to
β-actin. The intra-assay variability was <7%.
Data were analyzed with the LightCycler analysis software. No significant
cytotoxicity was observed using the trypan blue exclusion test of cell viability
for the macrophage cells when treated with any of the ash extracts at the level
of 1 μL extract to 1 mL medium (
