The full length of actin was amplified by RT-PCR in order to evaluate the synthesized cDNA by intact RNA. The presence of 138 bp bands confirmed the cDNA quality (figure 7). Figure 7 RT-PCR analysis of mRNA isolated from snap-frozen pancreatic tissues immersed in RNA-later for 24 h at -80ºC that were extracted with TriPure reagent. Lane M represents 50 bp DNA markers and lanes 1-5 show β-actin (138 bp) cDNAs amplified … Discussion Obtaining high-quality RNA is the first and most critical step in many molecular techniques such as reverse Inhibitors,research,lifescience,medical transcription real-time PCR (RT-qPCR), transcriptome

analysis that uses next-generation sequencing, array analysis, northern analysis, and cDNA library construction. Inhibitors,research,lifescience,medical RNA extraction is complicated because of the presence of ribonuclease enzymes in cells and tissues that can rapidly degrade RNA.14,17,18 RNases act without cofactors and are stable enzymes. The inactivation of RNases is difficult. Small amounts of RNase are sufficient to destroy RNA. In order to inactivate any RNases prior to surgery and in cases of removal of rat pancreatic tissue from the abdominal cavity, all surgical instruments should be cleaned with strong detergents, thoroughly rinsed, Inhibitors,research,lifescience,medical and placed in an oven for at

least 4 h at 240ºC. The place of surgery should be sterilized with NaOH and mild bleach to inactive the RNases.19 The pancreas has an extremely high level of RNase. RNA degradation occurs while the pancreas tissue is removed during dissection. In order

to increase efficiency dissection should be performed as quickly as possible.18,19 The pancreas is one of the important tissues that functions in the body’s homeostatic mechanisms. Therefore, improvement Inhibitors,research,lifescience,medical of RNA extraction procedures from the pancreas increases understanding of active pathways such as glucose regulation and redifferentiated β islets of the pancreas before and after treatment with different drugs. Knowledge of gene Inhibitors,research,lifescience,medical expression assays may potentially lead to the development of therapeutic drugs to restore β-cells and prevent apoptosis in diabetics.20 To identify novel metabolic genes and pathways that may play a role in the pathogenesis or treatment of diabetes, differential expression of metabolic genes is necessary. However, RNA extraction from to pancreatic tissue is difficult because of the abundance of RNase.21-23 Therefore we have attempted to design an efficient, simple optimized method to extract RNA from the pancreas based on our laboratory facilities. In order to enhance and correct this method, we assessed different common RNA extraction methods from pancreatic tissue with particular focus on the effect of frozen storage and RNase inhibition strategies, both of which affect RNA quality. The duration of surgery and amount of www.selleckchem.com/btk.html collected pancreatic tissue are the most crucial steps for obtaining intact RNA. Recent studies have shown a positive correlation between RNA degradation and the amount of pancreatic tissue.