Mm01205647 g1

Total RNA was extracted from INS-1D cells or mouse pancreatic islets using the RNeasy Mini Kit (Qiagen, Cologne, Germany). cDNA was prepared from 1 μg of total RNA using reverse transcriptase (SuperScript IIITM, Invitrogen) according to the manufacturer’s instructions. Quantitative PCR amplification was performed and analyzed using the TaqMan universal PCR master mix core reagent kit on StepOnePlus (Applied Biosystems, Foster, CA). The mRNA levels were evaluated with reference to beta actin (Actb) expression using the 2ΔΔCt method^{42 (link)}. TaqMan gene expression assay probes for rat Necab1 and Actb were obtained from Applied Biosystems (catalogue numbers Rn00574261_m1 and 4352931E, respectively, Applied Biosystems, Foster, IN). TaqMan gene expression assay probes for mouse Necab1, Actb, Insulin 1, and Insulin 2 were obtained from Applied Biosystems (Mm00724274_m1, Mm01205647_g1, Mm01278327_m1, and Mm03038438_m1, respectively).

RNA was extracted from snap frozen mouse liver tissue using the RNeasy Mini kit and RNase free DNase set according to the manufacturer’s recommendations (Qiagen GmbH, Düsseldorf, Germany), and RNA quantity and quality were analyzed with Nanodrop and gel. For quantification RNA was reverse transcribed using Taqman® reverse transcription reagents from Applied Biosystems (Invitrogen, Carlsbad, CA, USA). The resulting cDNA was used as template in real time reactions on a StepOne platform using the Taqman® Gene Expression assays Mm00518001_m1, Mm00841715_m1, Mm01205647_g1, and Mm03302249_g1, Mm00481849_m1, Mm00445911_m1, Mm00445961_m1, Mm00481216_m1, Mm00504941_m1, Mm00458272_m1, Mm00432638_m1, Mm01259143_g1 from Applied Biosystems (Invitrogen, Carlsbad, CA, USA). Expression values were normalized against the housekeeping gene Gapdh (Mm9999915_g1).