Type I interferon protects neurons from prions in in vivo models

Ishibashi, Daisuke; Homma, Takujiro; Nakagaki, Takehiro; Fuse, Takayuki; Sano, Kazunori; Satoh, Katsuya; Mori, Tsuyoshi; Atarashi, Ryuichiro; Nishida, Noriyuki

After prion infection, total RNA was harvested from the cells and brain tissues using a GenElute TM Mammalian Total RNA Miniprep kit (Sigma), according to the manufacturer's protocol. RNA was reverse-transcribed with SuperScript VILO (Invitrogen), and the resulting cDNA was used in a quantitative PCR through reaction with LightCycler Õ 480 SYBR Green I Master (Roche Applied Science) and the original primers listed below, and measured by a Light Cycler 480 instrument (Roche Applied Science). Murine Irf3 (NCBI accession number: NM_016849) cDNA was amplified using 5 0 -ACT GAA AAC CGT GGA CTT GC-3 0 as the sense primer and 5 0 -AGT CCA TGT CCT CCA CCA AG-3 0 as the antisense primer; murine Irf7 (NCBI accession number: NM_016850) cDNA, using 5 0 -GGT ATG CCG CCA AAT CTA AA-3 0 and 5 0 -GCT GAG GTC CAA AAT TTC CA-3 0 ; murine Tlr3 (NCBI accession number: NM_126166) cDNA, using 5 0 -AGC TTT GCT GGG AAC TTT CA-3 0 and 5 0 -GAA AGA TCG AGC TGG GTG AG-3 0 ; murine Tlr4 (NCBI accession number: NM_021297) cDNA, using 5 0 -CAG CAA AGT CCC TGA TGA CA -3 0 and 5 0 -AGA GGT GGT GTA AGC CAT GC-3 0 ; murine Tlr7 (NCBI accession number: NM_001290755) cDNA, using 5 0 -GGT ATG CCG CCA AAT CTA AA-3 0 and 5 0 -GCT GAG GTC CAA AAT TTC CA-3 0 ; murine retinoic acid-inducible gene 1 (Ddx58/RIGI, NCBI accession number: NM_172689) cDNA, using 5 0 -AAG GCT GAT GAG GAT GAT GG-3 0 and 5 0 -TGG TTT CAA TGG GCT GTG TA-3 0 ; murine melanoma differentiation-associated gene 5 (Ifih1/MDA5, NCBI accession number: NM_ 027835) cDNA, using 5 0 -GCT GCT AAA GAC GGA AAT CG-3 0 and 5 0 -TCT TGT CGC TGT CAT TGA GG-3 0 ; murine Ifnb (NCBI accession number: NM_010510.1) cDNA, using 5 0 -CCC TAT GGA GAT GAC GGA GA-3 0 and 5 0 -CTG TCT GCT GGT GGA GTT CA-3 0 , respectively. As an internal standard, mouse b-actin/Actb (NCBI accession number: NM_007393) cDNA sense primer, 5 0 -AAA TCG TGC GTG ACA TCA AA-3 0 and antisense primer, 5 0 -AAG GAA GGC TGG AAA AGA GC-3 0 , were used . The primers of Irf3, Irf7, Tlr3, Tlr7, and Ifih1/ MDA5 were designed to amplify the ORF region, and those of Tlr4, Ddx58/RIGI, Ifnb, and Actb were designed to amplify the ORF region across the intron. The results were normalized for the Actb mRNA level.