Author/Authors :
Dastgheib، Sanaz نويسنده Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran , , Irajie، Cambyz نويسنده Department of Resource Development and Management, Shiraz University of Medical Sciences, Shiraz, Iran , , Assaei، Raheleh نويسنده Department of Physiology, Faculty of Medicine, Lorestan University of Medical Sciences, Khoramabad, Iran Assaei, Raheleh , Koohpeima، Farhad نويسنده Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran , , Mokarram، Pooneh نويسنده Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran ,
Abstract :
Background: Optimized RNA extraction from tissues and cell lines consists of four main stages regardless of the method of extraction: 1) homogenizing, 2) effective denaturation of proteins from RNA, 3) inactivation of ribonuclease, and 4) removal of any DNA, protein, and carbohydrate contamination. Isolation of undamaged intact RNA is challenging when the related tissue contains high levels of RNase. Various technical difficulties occur during extraction of RNA from pancreatic tissue due to spontaneous autolysis. Since standard routine protocols yield unacceptable results in pancrease, we have designed a simple method for RNA extraction by comparing different protocols.
Methods: We obtained 20-30 mg pancreatic tissues in less than 2 min from 30 rats. Several methods were performed to extract RNA from pancreatic tissue and evaluate its integrity. All methods were performed three times to obtain reproducible results.
Results: Immersing pancreatic tissue in RNA-later for 24 h at -80?C yielded high quality RNA by using the TriPure reagent which was comparable to the commercial RNeasy Micro Kit. The quality of RNA was evaluated by spectrophotometer, electrophoresis and RT-PCR. We separated intact 28S and 18S ribosomal RNA (rRNA) when our procedure was compared with the RNeasy Micro Kit. Finally, full length of the actin gene was amplified by RT-PCR.
Conclusion: We designed a simple, fast, cost-effective method for complete RNA extraction from the least amount of quantitatively intact pancreatic tissue.
