Toni, Lee S.
(Department of Medicine, Division of Cardiology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States)
,
Garcia, Anastacia M.
(Department of Pediatrics, Division of Cardiology, University of Colorado Anschutz Medical Campus, Children’s Hospital Colorado, Aurora, CO, United States)
,
Jeffrey, Danielle A.
(Department of Medicine, Division of Cardiology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States)
,
Jiang, Xuan
(Department of Medicine, Division of Cardiology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States)
,
Stauffer, Brian L.
(Department of Medicine, Division of Cardiology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States)
,
Miyamoto, Shelley D.
(Department of Pediatrics, Division of Cardiology, University of Colorado Anschutz Medical Campus, Children’s Hospital Colorado, Aurora, CO, United States)
,
Sucharov, Carmen C.
(Department of Medicine, Division of Cardiology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States)
Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in sig...
Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis.•Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.•Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity.•In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.
Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis.•Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.•Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity.•In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.
1 Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction Anal. Biochem. 162 1987 156 159 Internet, cited 2018 May 3, Available from: http://www.ncbi.nlm.nih.gov/pubmed/2440339 2440339
2 Chomczynski P. Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on Nat. Protoc. 1 2006 581 585 Internet, cited 2018 May 3, Available from: http://www.ncbi.nlm.nih.gov/pubmed/17406285 17406285
3 Deng M.Y. Wang H. Ward G.B. Beckham T.R. McKenna T.S. Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription–PCR J. Vet. Diagn. Invest. 17 2005 574 578 Internet, cited 2018 Feb 23, Available from: http://www.ncbi.nlm.nih.gov/pubmed/16475517 16475517
4 Santiago-Vazquez L.Z. Ranzer L.K. Kerr R.G. Comparison of two total RNA extraction protocols using the marine gorgonian coral pseudopterogorgia elisabethae and its symbiont Symbiodinium sp Electron. J. Biotechnol. 9 2006 Internet, cited 2018 Feb 23, Available from: http://www.ejbiotechnology.info/content/vol9/issue5/full/15/index.html
5 Xiang X. Qiu D. Hegele R.D. Tan W.C. Comparison of different methods of total RNA extraction for viral detection in sputum J. Virol. Methods 94 2001 129 135 Internet, cited 2018 Feb 23, Available from: http://www.ncbi.nlm.nih.gov/pubmed/11337047 11337047
6 Sucharov C.C. Dockstader K. Nunley K. McKinsey T.A. Bristow M. β-Adrenergic receptor stimulation and activation of protein kinase A protect against α1-adrenergic-mediated phosphorylation of protein kinase D and histone deacetylase 5 J. Card Fail. 17 2011 592 600 Internet, cited 2016 Apr 5, Available from: http://www.ncbi.nlm.nih.gov/pubmed/21703532 21703532
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