Abstract
The majority of tumors, including melanoma, are phenotypically heterogeneous in that they contain various cell populations with differential expression of cell surface antigens such as CD133/Prominin-1. We have used fluorescence-activated cell sorting (FACS) technology to purify CD133+ and CD133− cellular subsets from mouse melanoma models for high-quality total RNA practical for downstream applications such as expression profiling. Implementation of this strategy can lead to higher resolution of transcripts that are potentially important for the survival and functionality of one cancer cell population relative to another. Suboptimal extraction of RNA after FACS is common and can ultimately result in misinterpretations that impede the effective design of novel therapies. Here, we describe a number of methods that have been amenable to the successful isolation of high-quality total RNA after FACS of CD133+ and CD133− mouse melanoma cell fractions.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Gershon D (2005) DNA microarrays: more than gene expression. Nature 437:1195-1198
Rappa G, Fodstad O, Lorico A (2008) The stem cell-associated antigen CD133 (Prominin-1) is a molecular therapeutic target for metastatic melanoma. Stem Cells 26:3008-3017
Zabierowski SE, Herlyn M (2008) Melanoma stem cells: the dark seed of melanoma. J Clin Oncol 26:2890-2894
Klein WM, Wu BP, Zhao S, Wu H, Klein-Szanto AJ, Tahan SR (2007) Increased expression of stem cell markers in malignant melanoma. Mod Pathol 20:102-107
Mizrak D, Brittan M, Alison MR (2008) CD133: molecule of the moment. J Pathol 214:3-9
Barrett MT, Glogovac J, Prevo LJ, Reid BJ, Porter P, Rabinovitch PS (2002) High-quality RNA and DNA from flow cytometrically sorted human epithelial cells and tissues. Biotechniques 32:888-896
Mack E, Neubauer A, Brendel C (2007) Comparison of RNA yield from small cell populations sorted by flow cytometry applying different isolation procedures. Cytometry A 71:404-409
D’Alessio G, Riordan JF (1997) Ribonucleases: structures and functions. Academic, San Diego, CA
Beintema JJ (1998) Introduction: the ribonuclease A superfamily. Cell Mol Life Sci 54:763-765
Bosenberg M, Muthusamy V, Curley DP, Wang Z, Hobbs C, Nelson B, Nogueira C, Horner JW, Depinho R, Chin L (2006) Characterization of melanocyte-inducible Cre recombinase transgenic mice. Genesis 44:262-267
Introduction to flow cytometry: a learning guide (2002) Becton, Dickinson and Company. 11-11032-03 rev. A
TRIzol reagent and TRIzol LS reagent technical note. Invitrogen Corp. Carlsbad, California
RNeasy® Micro Kit handbook (2007) QIAGEN sciences. Germantown, MD
QIAvac® 24 Plus handbook (2005) QIAGEN sciences. Germantown, MD
Qubit™ fluorometer instruction manual (2007) Invitrogen Corp. Carlsbad, California
Quant-iT™ RiboGreen RNA assay kit. Invitrogen Corp. Carlsbad, California
Kuschel M, Ausserer W (2000) Characterization of RNA quality using the Agilent 2100 Bioanalyzer. Agilent Technologies Application Notes
RNAlater® handbook (2006) QIAGEN sciences. Germantown, MD
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Tighe, S., Held, M.A. (2010). Isolation of Total RNA from Transgenic Mouse Melanoma Subsets Using Fluorescence-Activated Cell Sorting. In: Chittur, S.V. (eds) Microarray Methods for Drug Discovery. Methods in Molecular Biology, vol 632. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-663-4_2
Download citation
DOI: https://doi.org/10.1007/978-1-60761-663-4_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-662-7
Online ISBN: 978-1-60761-663-4
eBook Packages: Springer Protocols