Genetic and Functional Assessment of the Role of the rs13431652-A and rs573225-A Alleles in the G6PC2 Promoter that Strongly Associate With Elevated Fasting Glucose Levels

Genotype/expression correlation in human pancreatic cDNAs. Isolated pancreatic islets were prepared by collagenase digestion and density gradient purification (1; 2). After isolation, islets were cultured free-floating in M199 culture medium (Sigma-Aldrich) at 5.5 mmol/l glucose concentration. Within 3 days of isolation, the islets were used for RNA extraction. Total RNA was extracted from the islets using the RNeasy Protect Mini Kit (QIAGEN) and quantified by absorbance at A260/A280 in a PerkinElmer spectrophotometer (to ensure integrity of RNA. The ratio between absorbance at 260 nm [A260] and absorbance at 280 nm [A280] was higher than 1.65) and its integrity was assessed after electrophoresis in 1.0% agarose gels by ethidium bromide staining. Human mRNA levels were quantified, following reverse transcription, by real-time quantitative RT-PCR (3). We used genespecific probes and primer pairs for G6PC2 (ID: Hs01549773_m1) (Applied Biosystems). Each sample was run in duplicate, and the transcript quantity was normalized to the mRNA level of POLR2A (ID: Hs00172187_m1).