Qpcr primer assays

Liver and colon samples were stored at −80°C until RNA and DNA isolation using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Germany) according to the manufacturer's protocol. Concentration, respectively, purity was controlled with a Picodrop100 (Picodrop, UK). Complementary DNA (cDNA) was synthesized by reverse transcription using RT₂ First Strand Kit (Qiagen, Germany). cDNA was analyzed with real-time polymerase chain reaction (PCR) using qPCR Primer Assays (Qiagen, Germany) and RT₂ SYBR Green Master Mix (Qiagen, Germany) according to manufacturer's protocol. PCR conditions were as follows: initial step of 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min, ending with melting curve analysis (gradient melting of the products was performed at 0.5°C/10 s from 65°C to 95°C). Each sample was analyzed in duplicate with normalization to the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

Colon and liver samples were stored at −80 °C. RNA and DNA were isolated from liver and colon using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol. The concentration were measured respectively purity controlled with a Picodrop100 (Picodrop, Hinxton, UK). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA by reverse transcription using RT² First Strand Kit (Qiagen, Hilden, Germany). cDNA was analyzed in real-time PCR using qPCR Primer Assays (Qiagen, Hilden, Germany) and RT² SYBR Green Mastermix (Qiagen, Hilden, Germany) according to protocol. PCR conditions were as follows: initial step of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min, ending with melting curve analysis (gradient melting of the products was performed at 0.5 °C/10 s from 65 °C to 95 °C). Each sample was analyzed in duplicate, with normalization to the housekeeping gene glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) as an internal control.