Taqman universal pcr master mix core reagent kit

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).

Total RNA was extracted from isolated islets using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). After quantifying the RNA using spectrophotometry, 2.5 µg of RNA was heated at 85 °C for 3 min and then reverse-transcribed in a 25-µL reaction mixture containing 200 units of Superscript II RNase H-RT (Thermo Fisher Scientific), 50 ng random hexamers (Thermo Fisher Scientific), 160 µmol/l dNTP and 10 nmol/l dithiothreitol. The reaction mixture was incubated for 10 min at 25 °C, 60 min at 42 °C and 10 min at 95 °C^{26 (link)}.
Quantitative PCR amplification of cDNA from mouse islets was performed using a TaqMan universal PCR master mix core reagent kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). PCR was performed for 40 cycles, and the reactions were incubated for 2 min at 50 °C and 10 min at 95 °C for the initial steps. In each cycle, denaturation was performed for 15 s at 95 °C, and annealing/extension was performed for 1 min at 60 °C. PCR was performed in 20 µL of solution using cDNAs synthesized from 1.11 ng of total RNA. The amount of mRNA was normalized by dividing the amount of the mRNA of interest by that of Gapdh mRNA. Primers specific for mouse Ins1, Ins2, Pdx1, Nkx2.2, MafA and Gapdh were purchased as Assays-on-Demand Gene Expression Products (Applied Biosystems)^{26 (link)}.