Moloney murine leukemia virus reverse transcription kit

Cellular RNA was isolated by Trizol-Reagent according to the manufacturer’s instructions. Briefly, the DNA was removed from the samples using DNase treatment (DNA-free kit; Ambion Applied Biosystems) and cDNA was synthesized from the purified RNA using Moloney murine leukemia virus reverse transcription kit (Promega). Gene-specific primer sets are listed in Table 1 and Actin primer sets are used to produce a normalization control. Real-time PCR was carried out in triplicate with the SYBR Green PCR Master Mix (Applied Biosystems) and a 7900HT Fast Real-Time PCR machine (Applied Biosystems).

RNA from enriched IECs was isolated with an RNeasy minikit (Qiagen) by following the manufacturer’s instructions. RNA was treated with RQ1 RNase-free DNase (Promega), and subsequently, cDNA was synthesized using a Moloney murine leukemia virus reverse transcription kit (Promega). Quantitative RT-PCR was performed using Fast SYBR green master mix (Thermo Fischer Scientific) and different primer pairs (see Table S2 in the supplemental material).

RNA was isolated from purified IECs using the RNeasy minikit (Qiagen) according to the manufacturer’s instructions. RNA was treated with RQ1 RNase-free DNase (Promega) for 30 min, and subsequently cDNA was synthesized using a Moloney murine leukemia virus reverse transcription kit (Promega). Amplification from cDNA was performed using Power SYBR green PCR master mix (ThermoFisher Scientific). Assay reactions were performed in a 20-μl volume with 2× Power SYBR green PCR master mix with efficiency-optimized primers to a final concentration of 0.05 μM each and 1 to 10 ng total cDNA. All reactions were carried out in technical duplicates. The ΔΔC_t method (where C_t is threshold cycle) of quantification was performed to give the log₂ fold change from the expression of averaged baseline measurements (42 (link)). Expression was normalized to that of the housekeeping gene Gapdh. Primers used are listed in Table S3.