Abstract
Present-day techniques allow for massively parallel and high-throughput characterization of the somatic mutation status of samples. Most of these assays rely on whole specimen extracts, where heterogeneous spatial context of the specimen is lost. This chapter describes an up-to-date protocol for multiplexed, in situ genotyping of RNA in preserved tissue and cell lines, using padlock probes and rolling circle amplification. The presented approach allows for automated quantification of mRNA expression and mutation status, in single cells or in designated specimen areas. Briefly, mRNA is first reverse-transcribed to cDNA. Padlock probes specifically hybridize to the cDNA copy of the allele and become circularized and thereby physically linked to their targets. Following this conversion, padlock probes are copied in situ by rolling circle amplification and labeled with flourophore-conjugated probes, allowing for their detection with conventional fluorescence microscopy.
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
Similar content being viewed by others
References
Hanahan D (2014) Rethinking the war on cancer. Lancet 383:558–563
Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674
Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280:585–590
Lubeck E, Cai L (2012) Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat Methods 9:743–748
Player AN, Shen LP, Kenny D et al (2001) Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J Histochem Cytochem 49:603–612
Choi HMT, Beck VA, Pierce NA (2014) Next-generation in situ hybridization chain reaction: Higher gain, lower cost, greater durability. ACS Nano 8:4284–4294
Nilsson M, Malmgren H, Samiotaki M et al (1994) Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265:2085–2088
Nilsson M, Banér J, Mendel-Hartvig M et al (2002) Making ends meet in genetic analysis using padlock probes. Hum Mutat 19:410–415
Fire A, Xu SQ (1995) Rolling replication of short DNA circles. Proc Natl Acad Sci U S A 92:4641–4645
Banér J, Nilsson M, Mendel-Hartvig M, Landegren U (1998) Signal amplification of padlock probes by rolling circle replication. Nucleic Acids Res 26:5073–5078
Stenberg J, Nilsson M, Landegren U (2005) ProbeMaker: an extensible framework for design of sets of oligonucleotide probes. BMC Bioinformatics 6:229
Luo J, Bergstrom DE, Barany F (1996) Improving the fidelity of Thermus thermophilus DNA ligase. Nucleic Acids Res 24:3071–3078
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
Grundberg I, Kiflemariam S, Mignardi M et al (2013) In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics. Oncotarget 4:2407–2418
Carpenter AE, Jones TR, Lamprecht MR et al (2006) Cell Profiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7:R100
Petersen M, Wengel J (2003) LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol 21:74–81
Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3-new capabilities and interfaces. Nucleic Acids Res 40:1–12
Wolf B, Lesnaw JA, Reichmann ME (1970) A mechanism of the irreversible inactivation of bovine pancreatic ribonuclease by diethylpyrocarbonate. A general reaction of diethylpyrocarbonate. A general reaction of diethylpyrocarbonate with proteins. Eur J Biochem 13:519–525
Acknowledgements
We thank Evangelia Darai for conducting the A549/ONCO-DG1 genotyping experiment and providing images shown in Fig. 3.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Krzywkowski, T., Hauling, T., Nilsson, M. (2017). In Situ Single-Molecule RNA Genotyping Using Padlock Probes and Rolling Circle Amplification. In: White, S., Cantsilieris, S. (eds) Genotyping. Methods in Molecular Biology, vol 1492. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6442-0_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6442-0_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6440-6
Online ISBN: 978-1-4939-6442-0
eBook Packages: Springer Protocols