Rotor gene q 2plex hrm real time pcr system

Real-time RT-PCR was performed for TNF-α, IFN-γ, IL-1β, and beta-2-microglobulin as the housekeeping gene. Liver samples were harvested after IT and stored until use. Total mRNA was extracted using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. cDNA was generated using a QuantiTect Reverse Transcription Kit (Qiagen) and amplified with a Rotor-Gene Q 2PLEX HRM Real-Time PCR system (Qiagen). TNF-α, IFN-γ, and IL-1β expressions were investigated using appropriate primers and probes (Taqman Universal PCR MasterMix, TaqmanGene Expression Assays, Applied Biosystems) with 32 ng of reverse transcribed total RNA in a total volume of 25 µl (Supplementary Table S1). The amplification protocol consisted of denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 10 s.

Complementary DNA was generated using a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) and amplified using a Rotor-Gene Q 2PLEX HRM Real-Time PCR system (Qiagen). The quantitative PCR reactions, which were prepared to a final volume of 25 μl, included 2 × Rotor-Gene SYBR Green PCR Mix (Qiagen), 10 μmol/l forward/reverse primers, and 32 ng of each complementary DNA sample. The amplification protocol involved denaturation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 5 s and 60 °C for 10 s. Beta-2-microglobulin (B2M) was used as an internal control. The primers used are shown in 'Supplementary Information 2'.