Semi-quantitative RT-PCR on amplified cDNA from sorted cells validates I-cells enriched mRNA transcripts.

Semi-quantitative RT-PCR analysis using amplified cDNA from eGFP+ and eGFP− cells. Left panel: eGFP+ cells were highly enriched in mRNA transcripts encoding eGFP and CCK whereas these transcripts were not detected in eGFP− cells. eGFP− cells were enriched in mRNA transcripts encoding AKP3 and MUC2, markers of enterocytes and goblet cells respectively. Right panel: Validation of Gpr40/Ffar1, Gpr120/O3far1, Gpr41/Ffar3, Gpr43/Ffar2, Gpr119 and CB1 mRNA transcripts enrichment in I-cells. 18S rRNA was used as a loading control. PCR products were amplified simultaneously, using different number of cycles (low-for lower number of cycles and high-for higher number of cycles). Equal volumes of each reaction were run on the same 2% agarose gel. L declares Hyperladder V (50–250 bp, Bioline, UK). Asterisk indicates primer dimers.