Isogen 2 isolation reagent

Total RNA was extracted from the resected mouse organs using ISOGEN II Isolation Reagent (Nippon Gene, Tokyo, Japan). Reverse transcription was then performed using ReverTraAce qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). A real-time polymerase chain reaction (PCR) was performed using FastStart SYBR^® Green Master Mix (Roche Applied Science, Penzberg, Germany) with specific primers on a StepOnePlus™ Realtime PCR system (Applied Biosystems, Foster City, CA, USA). The primers used are shown in Table 2.

ISOGEN II Isolation Reagent was used to extract total RNA from all cell types prepared in this study (Nippon Gene, Tokyo). Reverse transcription was then performed using ReverTraAce qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Real-time polymerase chain reaction (PCR) was performed using FastStart SYBR^® Green Master Mix (Roche Applied Science, Penzberg, Germany) with specific primers on a StepOnePlus™ Real-time PCR system (Applied Biosystems, Foster, CA).
The primers used were as follows: ACTB (forward: 5′-ctggaacggtgaaggtgaca-3′; reverse: 5′-aagggacttcctgtaacaatgca-3′); REIC/DKK3 (forward: 5′-aactgatggaggacacgcag-3′; reverse: 5′-gctgggaggtaagtttgcca-3′); CD274 (PD-L1) (forward: 5′-ggcatccaagatacaaactcaa-3′; reverse: 5′-cagaagttccaatgctggatta-3′); C5AR (forward: 5′-ggagggaccttcgatcctc-3′; reverse: 5′-ggggtggtataattgaaggagtt-3′); CXCR2 (forward: 5′-gaggcacagtgaagacatcg-3′; reverse: 5′-gctgggcttttcacctgtag-3′); CXCR6 (forward: 5′-ggggatgacatgtgactcctat-3′; reverse: 5′-cgtgctcacctcttcaacct-3′); ACKR3 (CXCR7) (forward: 5′-cagttgttgcaaagtgctcag-3′; reverse: 5′-cgggcaatcaaatgacct-3′); and CMTM6 (forward: 5′-ggacttcagctgagattgctg-3′; reverse: 5′-ccctagtggtattttcaggttttc-3′).