Reverse transcriptase buffer

RNA was extracted from islet using the RNeasy micro kit (Qiagen, Valencia, CA, USA) and reverse transcribed (1 μg total RNA, 1 μL random primer (50 μmol/L; Applied Biosystems, Foster City, CA, USA), 1× reverse transcriptase buffer and 10 units reverse transcriptase (Promega, Madison, WI, USA) in a total volume of 20 μL). The RNA and primer were heated to 72 °C and slowly cooled before reverse transcription at 42 °C for one hour. The room temperature reaction was then diluted to 100 μL with RNase-free water. For real-time PCR analysis, 2.5% of the total room temperature reaction was used as input for PCR using SYBR Green Master Mix (Applied Biosystems) on an ABI 7900 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Primers for cyclin D1, cyclin D2, cyclin D3, Cdk4, GLUT-2, GCK, Foxo-1, PDX-1 and Ngn3 were previously described [17 ,41 (link)]. For analysis of Ngn3 mRNA abundance, RNA was extracted from the remnant pancreas, and for analysis of other genes, RNA was extracted from islets [41 (link)].

RPMI-1640 culture solution, foetal calf serum (Gibco, Gaithersburg, MD, USA), Ox-LDL, HMCs (Basic Institute of Peking Union Medical College, Beijing, China), IL-1β, LOX-1 antibody (R&D, Abingdon, UK), and Dil-Ox-LDL (Biomed Technologies, Mt. Arlington, NJ, USA) were used. A polymerase chain reaction (PCR) system, reverse transcriptase buffer (Promega, Madison, WI, USA), dNTPs, and Oligo(dT) primers (Sangon Biotech, Shanghai, China) were used. The primer sequences were as follows: LOX‑1 forward, 5'-ACAGAGGCCATTCCGAAATCA-3'; reverse, 5'-GGTAGAGTCTGGAGATGGACCACA-3'; GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′; reverse, 5′-ATGGTGGTGAAGACGCCAGT -3′.