Luna universal qrt pcr master mix

Total RNA from organ or cell lysates was isolated and purified using RNeasy Plus Micro Kit. Following the manufacturer’s instructions, cDNA was prepared with the high-capacity cDNA reverse transcription kit (Applied Biosystems). The list of gene targets and catalog numbers used throughout the study can be found in Table S1. Singleplex qRT–PCR protocol was performed using Luna Universal qRT-PCR Master Mix (New England Biolabs). Target gene expression cycle thresholds were normalized to the housekeeping gene and calculated into relative expression using the 2^−ΔΔCq method (73 (link)).

Table S1 RT–qPCR reagent information used throughout the study. Predesigned probes and customized primer/probe sets for mRNA gene expression assays.

To confirm the expression of selected genes of interest involved in fatty acid uptake regulation, RT-qPCR experiments were performed. Fully differentiated Caco-2 cells were starved for 60 min with serum-free medium followed by incubation with the test compounds for 30, 60 and 90 min at 37 °C. Afterwards, RNA was isolated using Monarch® Total RNA Miniprep Kit (New England Biolabs) and reverse-transcribed to cDNA using LunaScript® RT SuperMix Kit (New England Biolabs) according to the manufacturer's protocol. PCR was subsequently performed using Luna® Universal qRT-PCR Master Mix (New England Biolabs) on a Step-One Plus Device (Applied Biosystems, Thermo Fisher Scientific, Austria). The primer pairs used during the PCR can be found in Table S1. Primers not previously reported, namely for DPP4, GCG, GLP1R, GIP and GIP-R, were validated by sequencing the obtained PCR product (Sanger Sequencing, Eurofins, Germany). Gene expression is given as fold change compared to the corresponding control. The starting concentrations of the respective mRNA used for reverse transcription were calculated using LinRegPCR Version 2021.2 and compared to the corresponding control after normalization to HPRT1 and GAPDH as reference genes.

cDNA was generated
from RNA (1.6 μg)
using SuperScript IV First-Strand Synthesis System (Invitrogen). Each
qRT-PCR reaction (20 μL) contained cDNA (5 ng) as template and
the appropriate primer pairs (0.4 μM; Table S1C). Samples were analyzed in technical duplicates. Amplicons
were detected with Luna Universal qRT-PCR Master Mix (New England
Biolabs) in a CFXConnect Real-Time PCR Instrument (Bio-Rad Laboratories). C_q values were calculated using LinRegPCR^{85 (link)} after correcting for amplicon efficiency. C_q values of technical duplicates were typically
within ±0.25 of each other. holB, which encodes
DNA polymerase III, was used as reference gene. Its transcription
levels were verified to remain constant in the experimental conditions
tested here.