Gene Expression Quantification by Real-Time PCR

The total RNA was synthesized into first-strand complementary DNA (cDNA) using a random primer (9-mer, Invitrogen, Carlsbad, CA, USA) and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Enzynomics Co., Daejeon, Republic of Korea) (Abdellaoui et al. 2023 (link)). The gene expression was quantified using TOPreal™ qPCR 2X PreMIX (Enzynomics Co.), the Eco Real-Time PCR system (Illumina, San Diego, CA, USA), and the following specific primers: mouse Interleukin (IL)-1β (Gene Bank ID, NM_008361, 133 bp), 5′-CCC AAG CAA TAC CCA AAG AA-3′ and 5′-GCT TGT GCT CTG CTT GTG AG-3′; mouse IL-6 (NM_031168, 102 bp), 5′-CCG GAG AGG AGA CTT CAC AG-3′ and 5′-TCC ACG ATT TCC CAG AGA AC-3′; mouse IL-10 (NM_010548, 142 bp), 5′-GCC TGG CTC AGC ACT GCT AT-3′ and 5′-GAA GGC AGT CCG CAG CTC TA-3′; mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh, NM_001289726, 223 bp), 5′-AAC TTT GGC ATT GTG GAA GG-3′ and 5′-ACA CAT TGG GGG TAG GAA CA-3′; human IL-1β (NM_000576, 180 bp), 5′-CTG TCC TGC GTG TTG AAA GA-3′ and 5′-TTC TGC TTG AGA GGT GCT GA-3′; human IL-6 (NM_000600, 175 bp), 5′-TAC CCC CAG GAG AAG ATT CC-3′ and 5′-TTT TCT GCC AGT GCC TCT TT-3′; human IL-10 (NM_000572, 253 bp), 5′-TGA GAA CAG CTG CAC CCA CT-3′ and 5′-GTT CAC ATG CGC CTT GAT GT-3′; human Gapdh (NM_001256799, 278 bp), 5′-ACT GGC GTC TTC ACC ACC AT-3′ and 5′-GGG CCA TCC ACA GTC TTC TG-3′.