Bio sybr green master mix

Total RNA of islets or cells was extracted by TRIzol reagent (Invitrogen, USA) according to the manufacturer's instruction. 0.5–1 μg total RNA was transcribed to complementary DNA (cDNA) using the cDNA ReverseTranscription kit (Cat. 4375575, Applied Biosystem, USA). One microliter cDNA was mixed with 0.25 μM forward primer, 0.25 μM reverse primer, 5 μl Bio SYBR Green Master mix (Takara, Japan) and dH₂O up to 10 μl. qPCR was performed in triplicate on Applied Biosystem 7500 fast real-time PCR system (Thermo Fisher Scientific, USA). To calculate the relative transcriptional expression, the Ct values of genes were normalized by average Ct values of GAPDH as ΔCt. The relative transcriptional expression of interested genes was indicated with 2^{(−−ΔΔCt)}. The sequences of forward and reverse primers were as follows: mouse Glucagon, 5′-TTC CCA GAA GAA GTC GCC ATT−3′, 5′- GGT GCT CAT CTC GTC AGA GAA−3′; mouse CFTR, 5′- GCT GAC ACT TTG CTT GCC CTG AG−3′, 5′- GCT TGC TGA TGG TCG ACA TAG GG−3′; mouse PC2, 5′- AGA GAG ACC CCA GGA TAA AGA TG−3′, 5′- CTT GCC CAG TGT TGA ACA GGT−3′, mouse GAPDH, 5′- AAC GAC CCC TTC ATT GAC−3′, 5′- TCC ACG ACA TAC TCA GCA C−3′.