Optimization of RNA Extraction from Rat Pancreatic Tissue

نویسندگان

  • Cambyz Irajie Department of Resource Development and Management, Shiraz University of Medical Sciences, Shiraz, Iran
  • Farhad Koohpeima Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
  • Pooneh Mokarram Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; and Gasteroenterohepatology Research Center, Nemazee Hospital, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran;and Faculty for Advanced Biomedical Sciences, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
  • Raheleh Assaei Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical Sciences, Shiraz, Iran
  • Sanaz Dastgheib Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran; and Student Research Committee, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
چکیده مقاله:

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 -80ºC 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.

برای دانلود باید عضویت طلایی داشته باشید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

optimization of rna extraction from rat pancreatic tissue

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 ...

متن کامل

RNA Extraction from Animal and Human\'s Cancerous Tissues: Does Tissue Matter?

The reliability of gene expression profiling based technologies and methods to find transcriptional differences representative for the original samples is influenced by the quality of the extracted RNA. Hence, RNA extraction is the first step to investigate the gene expression and its function. Consequently, the quality of extracted RNA is really significant. Correspondingly, this research was ...

متن کامل

Tissue homogenization as a key step in extracting RNA from human and rat pancreatic tissue.

1.Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts and J.D. Watson. 1989. Molecular Biology of the Cell, 2nd ed. Garland Publishing, New York. 2.Atlas, R.M. and L.C. Parks. 1993. Handbook of Microbiological Media, p. 766-767. CRC Press, Boca Raton. 3.Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Bi...

متن کامل

منابع من

با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

ذخیره در منابع من قبلا به منابع من ذحیره شده

{@ msg_add @}


عنوان ژورنال

دوره 39  شماره 3

صفحات  282- 288

تاریخ انتشار 2014-05-01

با دنبال کردن یک ژورنال هنگامی که شماره جدید این ژورنال منتشر می شود به شما از طریق ایمیل اطلاع داده می شود.

کلمات کلیدی

میزبانی شده توسط پلتفرم ابری doprax.com

copyright © 2015-2023