RNA extraction and quantitative real time polymerase chain
reaction (qRT-PCR)
Total RNA was extracted from peripheral blood, fresh-frozen biopsies and
pWSLV-02-LILRA3 plasmid- or pWSLV-02-transfected U937 cells using the
Trizol reagent (Invitrogen, Carlsbad, USA). The quantity and quality of
the RNA samples were assessed using a NanoDrop2000 spectrophotometer
(Thermo Scientific, Waltham, USA). Total RNA (1 μg) was used to
synthesize cDNA using a first-strand cDNA synthesis kit (Thermo
Scientific, Waltham, USA). qRT-PCR was subsequently performed using the
QuantStudioTM6 Flex Real-Time PCR instrument (ABI, Carlsbad, USA) with
the SYBR® Premix Ex TaqTM II mix (Takara, Kusatsu,
Japan). The 2−ΔΔCT method was applied to determine the
relative mRNA levels normalized to the housekeeping gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).