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The reaction mixture in total volume of 20 ��l was prepared containing: 4 ��l 5X Reaction Buffer, Tyrosine Kinase Inhibitor Library concentration 1 ��l RiboLock RNase Inhibitor (20 U/��l), 2 ��l 10 mM dNTP Mix, and 1 ��l RevertAid M-MuLV Reverse Transcriptase (200 U/��l). The prepared mixture incubated in a thermocycler device (Eppendorf Mastercycler, USA) for 5 minutes at 25��C followed by 60 minutes at 42��C. The reaction terminated by heating at 70��C for 5 minutes (according to the manufacturer��s instructions). Agarose gel electrophoresis (1%) was run to confirm the quality and integrity of the synthesized cDNA. The cDNA concentration and purity was determined using a NanoDrop UV spectrophotometer (BioTek Laboratories, Inc., USA). Real-Time Polymerase Chain Reaction (qPCR) SYBR Premix Ex Taq II (Tli Rnase H Plus), (TaKaRa, China) on CFX96 real-time PCR detection system (Bio-Rad, USA) was applied to amplify and determine mRNA expression levels of SNARE proteins.17 18S rRNA housekeeping gene was used as an internal control.18 The forward and reverse primers used for 18S rRNA were: 5`-dGTAACCCGTTGAACCCCATT and 5`-dCCATCCAATCGGTAGTAGCG. Gene-specific primers were designed using AlleleID7 software (Premier Biosoft Corporation, USA). To use in reaction, primers were prepared at a concentration of 10 ?M. Primer pairs properties are listed in table 1. qPCR reaction mixture was prepared according to the manufacturer��s instructions for each sample as follows: 10 ��l SYBR Premix Ex Taq II (1X), 1 ��l of each primer (0.4 ��M), 1 ��l cDNA template (Lonafarnib research buy cycling was performed in 40 cycles. Table1 Properties of primer pairs used in real-time RT-PCR Insulin Assay Rat Insulin (INS) ELISA Kit was used to detect the insulin levels. VAV2 This kit measured insulin levels in serum samples of rats based on double-antibody sandwich technique (according to supplier��s instruction). Insulin resistance index was calculated as follows: insulin (��U/ml)��glucose (mmol/L)/22.5.19 Statistical Analysis The obtained data are presented as mean��SD. In order to statistically compare data between different groups, one way ANOVA followed by post hoc Turkey��s test was used. P values