Abstract
Purification of intact RNA is the primary step of many molecular biology techniques, including Northern blotting, RNase protection, quantitative polymerase chain reaction, and microarray assays. RNA extraction is typically conducted using either a phenol-choloroform or a solid phase method. This article concentrates primarily on the former approach, which is highly versatile, and is easily adapted to different tissues ranging from whole organs down to submillimeter biopsy punches.
The major problem with RNA extraction is the ubiquitous nature of RNases, enzymes that rapidly degrade RNA. RNases may be difficult to eliminate, although with care and appropriate countermeasures, reproducible extraction of high-quality RNA from important biological samples should be attainable.
This article focuses on the isolation of RNA from the tissue collection step, homogenization all the way through to the quantification of the purified nucleic acid, providing guidelines for the prevention of the problems associated with RNAse contamination.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sambrook, J., and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Chomczynski, P., and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.
Eckhart, L., Bach, J., Ban, J., and Tschachler, E. (2000) Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity. Biochem. Biophys. Res. Commun. 271, 726–730.
Nir, I., and Agarwal, N. (1993) Diurnal expression of c-fos in the mouse retina. Mol. Brain Res. 19, 47–54.
Peirson, S. N., Butler, J. B., and Foster, R. (2003) Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res. 31, e73.
Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15, 532–534, 536-537.
Groppe, J. C., and Morse, D. E. (1993) Isolation of full-length RNA templates for reverse transcription from tissues rich in RNAse and proteoglycans. Anal. Biochem. 210, 337–343.
Re, P., Valhmu, W. B., Vostrejs, M., Howell, D. S., Fischer, S. G., and Ratcliffe, A. (1995) Quantitative polymerase chain reaction assay for aggrecan and link protein gene expression in cartilage. Anal. Biochem. 225, 356–360. Erratum in Anal. Biochem. 228, 358.
Schick, B. P., and Eras, J. (1995) Proteoglycans partially co-purify with RNA in TRI Reagent and can be transferred to filters by Northern blotting. Biotechniques 18, 574–576, 578.
Chomczynski, P., and Mackey, K. (1995) Short technical reports. Modification of the TRI reagent procedure for isolation of RNA from polysaccharide-and proteoglycan-rich sources. Biotechniques 19, 942–945.
Wilfinger, W. W., Mackey, K., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22, 474–476, 478–481.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Peirson, S.N., Butler, J.N. (2007). RNA Extraction From Mammalian Tissues. In: Rosato, E. (eds) Circadian Rhythms. Methods in Molecular Biology™, vol 362. Humana Press. https://doi.org/10.1007/978-1-59745-257-1_22
Download citation
DOI: https://doi.org/10.1007/978-1-59745-257-1_22
Publisher Name: Humana Press
Print ISBN: 978-1-58829-417-3
Online ISBN: 978-1-59745-257-1
eBook Packages: Springer Protocols