Total RNA was isolated from liver or ileum using Ribopure (Invitrogen, ThermoFisher Scientific, USA). Total RNA was subjected to DNase I treatment using Turbo DNA-free (Invitrogen, ThermoFisher Scientific, USA), and RNA integrity was confirmed by agarose gel electrophoresis. Afterwards, cDNA was synthesized by oligo(dT)-primed reverse transcription with Superscript II (Invitrogen, ThermoFisher Scientific, USA). qPCRs were performed using a CXF96® Touch (Bio-Rad, California, USA). The reaction solution was carried out in a volume of 20 μl, containing 10 pmol of both forward and reverse primers, 10x SYBR Premix Ex Taq (Takara Bio Inc., Japan) and the appropriate nanograms of the cDNA stock. Rps29 was used as a reference gene for qPCR. The primer sequences were obtained either from the Atlas RT-PCR Primer Sequences (Clontech, CA, USA) or designed using Primer3 software (University of Massachusetts Medical School, MA, USA) (Rozen & Skaletsky, 2000 (link)).
Samples were analysed in duplicate on each assay. Amplification of non-specific targets was discarded using the melting curve analysis method for each amplicon. qPCR efficiency and linearity were assessed by optimization of the standard curves for each target. The transcription was quantified with CFX Maestro 2.0 software (Bio-Rad, California, USA) using the efficiency correction method (Pfaffl, 2001 (link)).
Samples were analysed in duplicate on each assay. Amplification of non-specific targets was discarded using the melting curve analysis method for each amplicon. qPCR efficiency and linearity were assessed by optimization of the standard curves for each target. The transcription was quantified with CFX Maestro 2.0 software (Bio-Rad, California, USA) using the efficiency correction method (Pfaffl, 2001 (link)).