Evaluation and Optimization of Various Procedures of RNA Extraction from Human Frozen Tissues

Document Type : Original Article(s)

Authors

1 Division of Genetics, Department of Biomedical Sciences, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

2 Department of Genetics, School of Basic Sciences and Research Institute of Biotechnology, Shahrekord University, Shahrekord, Iran.

Abstract

Background: Utilizing an intact RNA is essential while performing molecular genetics methods like microarray and quantitative Real-time polymerase chain reaction (RT-PCR). Using RNA with poor quality may affect the results of downstream manipulations. Our aim in the current study was to compare and optimize various procedures for RNA extraction by using different kits and DNase treatment to find a standard method for RNA extraction from human frozen tissues.Methods: Using Cinnagen RNX plus solution, Qiagen RNeasy Mini Kit and Qiagen RNeasy Plus Mini Kit, total RNA was extracted from human frozen tissues comprising stomach (#40), glioma (#5) and breast (#15). For genomic DNA elimination, extracted RNA was treated with DNase enzyme. After RNA extraction, its quantity and quality was assessed by spectrophotometry and gel electrophoresis. After cDNA synthesis, RT-PCR technique by specific primers designed for TBP gene was performed and results were assessed by agarose gel electrophoresis.Finding: Extracted RNA by Cinnagen RNX plus solution did not show good results in RT-PCR after DNase treatment. Extracted RNAs by Qiagen RNeasy Mini Kit and Qiagen RNeasy Plus Mini Kit were of high quality for RT-PCR reaction because of using DNase treatment during the extraction procedure and an additional genomic DNA elimination column respectively.Conclusion: Combining Qiagen RNeasy Mini Kit, Qiazol solution and DNase treatment during the extraction procedure is the most suitable method for RNA extraction from human frozen tissues and the extracted RNA can be used with assurance in downstream molecular techniques.

Keywords

References

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Journal of Isfahan Medical School
Volume 29, Issue 133 - Serial Number 133
March and April 2011
Pages 327-335

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History

  • Receive Date: 16 February 2011
  • Revise Date: 01 November 2024
  • Accept Date: 06 April 2022

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