Q-PCR analyses for mouse cytokines
For transcriptional analyses, colon samples from DSS-treated mice were
homogenized in TRIzol (Thermo Fisher Scientific) and stored at −80°C
until further processed. RNA was initially extracted with chloroform and
then further purified by lithium chloride precipitation to remove DSS a
strong inhibitor of downstream PCR applications [23]. Reverse
transcription of total RNA was performed using the High-Capacity cDNA
Reverse Transcription kit according to the manufacturer’s
recommendations (Thermo Fisher Scientific). Quantification of expression
was detected on a StepOnePlus Real-Time PCR (Thermo Fisher Scientific)
using the following TaqMan Gene Expression Assays (Applied Biosystems):Il-1b (Mm00434228), Il-6 (Mm00446190), IL-17a(Mm00439618) and tnfa (Mm00443258). Gene expression was
normalized as n-fold difference to mouse Hprt1 (Mm00446968)
according to the cycling threshold.