Isolation and Analysis of Pancreatic Islets

Pancreatic islets were isolated from the pancreas by collagenase digestion as previously described [21 (link)]. Briefly, male mice at age 10 weeks were anesthetized with Sevoflurane (Mylan, Inc., Southpoint, PA, USA), followed by injection of collagenase into the common bile duct. The pancreas was removed by dissection and was incubated at 37°C for 12 min. The islets were then collected under a stereoscopic microscope. Total RNAs from islets were extracted with the Isogen RNA extraction kit (Nippon Gene, Tokyo, Japan), and cDNA was prepared with ReverTra Ace (Toyobo, Osaka, Japan). For quantification, gene-specific primers (S4 Table) were used with Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) in a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression level of each target gene was normalized to the level of Actb mRNA. Each experiment was performed in three biological replicates.