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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....

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Autores principales: Dastgheib, Sanaz, Irajie, Cambyz, Assaei, Raheleh, Koohpeima, Farhad, Mokarram, Pooneh
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Shiraz University of Medical Sciences 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027008/
https://www.ncbi.nlm.nih.gov/pubmed/24850986
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author Dastgheib, Sanaz
Irajie, Cambyz
Assaei, Raheleh
Koohpeima, Farhad
Mokarram, Pooneh
author_facet Dastgheib, Sanaz
Irajie, Cambyz
Assaei, Raheleh
Koohpeima, Farhad
Mokarram, Pooneh
author_sort Dastgheib, Sanaz
collection PubMed
description 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.
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spelling pubmed-40270082014-05-21 Optimization of RNA Extraction from Rat Pancreatic Tissue Dastgheib, Sanaz Irajie, Cambyz Assaei, Raheleh Koohpeima, Farhad Mokarram, Pooneh Iran J Med Sci Original Article 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. Shiraz University of Medical Sciences 2014-05 /pmc/articles/PMC4027008/ /pubmed/24850986 Text en © 2014: Iranian Journal of Medical Sciences This is an Open Access article distributed under the terms of the Creative Commons Attribution License, (http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Dastgheib, Sanaz
Irajie, Cambyz
Assaei, Raheleh
Koohpeima, Farhad
Mokarram, Pooneh
Optimization of RNA Extraction from Rat Pancreatic Tissue
title Optimization of RNA Extraction from Rat Pancreatic Tissue
title_full Optimization of RNA Extraction from Rat Pancreatic Tissue
title_fullStr Optimization of RNA Extraction from Rat Pancreatic Tissue
title_full_unstemmed Optimization of RNA Extraction from Rat Pancreatic Tissue
title_short Optimization of RNA Extraction from Rat Pancreatic Tissue
title_sort optimization of rna extraction from rat pancreatic tissue
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027008/
https://www.ncbi.nlm.nih.gov/pubmed/24850986
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