Rotor gene 6000 series software version 1

Spleen tissues from control and PsAE mice were used to obtain total RNA. It was isolated using TRIzol ® reagent (Invitrogen, 15596026) according to the manufacturer protocol. Total RNA was reverse transcribed, using random hexamer primers and Moloney murine leukemia virus retrotranscriptase (Invitrogen-Life Technologies, USA). Real-time qPCR was performed with cDNA samples and EVA Green (Biotium, Hayward, CA) using a Rotor-Gene 6000 Series Software version 1.7 (Corbett). The samples were amplified in triplicate. The oligonucleotide primers (OligoLTA, Thermo Fischer Scientific) used were: perforin (PRF-1) sense: 5′-GAGAAGACCTATCAGGACCA-3′ and antisense 5′-AGCCTGTGGTAAGCATG-3′; granzyme B (GRZ-B) sense: 5′-CCTCCTGCTACTGCTGAC-3′, and antisense: 5′-GTCAGCACAAAGTCCTCTC-3; and, HPRT1 (hypoxanthine-guanine phosphoribosyltransferase) sense: 5′-GTTGGATACAGGCCAGACTTTGTTG-3′, and antisense; 5′-GATTCAACTTGCGCTCATCTTAGGC-3’. PCR reactions were initiated with 5 min incubation at 95 °C, followed by 40 cycles of: 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s mRNA expression levels of PRF-1 and GRZ-B were normalized to the HPRT1 reference gene. To compare the relative expression between treatments, the mRNA levels were expressed as a ratio using the delta-delta method. Relative expression was calculated as 2^−ΔΔCt.