Abstract
Rapid development of high-throughput DNA analyzation methods has enabled global characterization of genetic landscapes and aberrations in study subjects in a time and cost effective fashion. In most methods, however, spatial tissue context is lost since sample preparation requires isolation of nucleic acids out of their native environment. We hereby present the most recent protocol for multiplexed, in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification. We take advantage of a single nucleotide variant within conserved ACTB mRNA to successfully differentiate human and mice cocultured cells and apply presented protocol to genotype PCDH X and Y homologs in human brain. We provide a method for automated characterization and quantitation of target mRNA in single cells or chosen tissue area. mRNA of interest, harboring a polymorphism, is first reverse-transcribed to cDNA. Allele specific padlock probes are hybridized to the cDNA target and enzymatically circularized maintaining a physical link with the parent mRNA molecule. Lastly, circularized probes are replicated in situ, using rolling circle amplification mechanism to facilitate detection.
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References
Kitpipit T, Tobe SS, Kitchener AC et al (2012) The development and validation of a single SNaPshot multiplex for tiger species and subspecies identification – implications for forensic purposes. Forensic Sci Int Genet 6:250–257
Huang C, Chang M, Huang M, Lee F (2011) Rapid identification of Lactobacillus plantarum group using the SNaPshot minisequencing assay. Syst Appl Microbiol 34:586–589
Diekstra A, Bosgoed E, Rikken A et al (2015) Translating Sanger-based routine DNA diagnostics into generic massive parallel ion semiconductor sequencing. Clin Chem 61:154–162
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 V, Pierce N (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
Petersen M, Wengel J (2003) LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol 21:74–81
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
Untergasser A, Cutcutache I, Koressaar T et al (2012) Primer3-new capabilities and interfaces. Nucleic Acids Res 40:1–12
Larsson C, Grundberg I, Söderberg O (2010) In situ detection and genotyping of individual mrna molecules. Nat Methods 7:395–397
Stenberg J, Nilsson M, Landegren U (2005) ProbeMaker: an extensible framework for design of sets of oligonucleotide probes. BMC Bioinformatics. doi:10.1186/1471-2105-6-229
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
Carpenter AE, Jones TR, Lamprecht MR et al (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7:R100
Johansson MM, Lundin E, Qian X et al (2016) Spatial sexual dimorphism of X and Y homolog gene expression in the human central nervous system during early male development. Biol Sex Differ 7:5
Larsson C, Koch J, Nygren A et al (2004) In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes. Nat Methods 1:227–232
Levin JD, Fiala D, Samala MF et al (2006) Position-dependent effects of locked nucleic acid ( LNA ) on DNA sequencing and PCR primers. Nucleic Acids Res 34:1–11
Wolf B, Lesnaw JA, Reichmann ME (1970) A mechanism of the irreversible inactivation of bovine pancreatic ribonuclease by diethylpyrocarbonate. Eur J Biochem 13:519–525
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
Ke R, Mignardi M, Pacureanu A et al (2013) In situ sequencing for RNA analysis in preserved tissue and cells. Nat Methods 10:857–860
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Krzywkowski, T., Nilsson, M. (2018). Padlock Probes to Detect Single Nucleotide Polymorphisms. In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_14
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DOI: https://doi.org/10.1007/978-1-4939-7213-5_14
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7212-8
Online ISBN: 978-1-4939-7213-5
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