Simple cDNA normalization using kamchatka crab duplex-specific nuclease
- PMID: 14973331
- PMCID: PMC373426
- DOI: 10.1093/nar/gnh031
Simple cDNA normalization using kamchatka crab duplex-specific nuclease
Abstract
We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.
Figures




Similar articles
-
Normalization of full-length-enriched cDNA.Methods Mol Biol. 2011;729:85-98. doi: 10.1007/978-1-61779-065-2_6. Methods Mol Biol. 2011. PMID: 21365485
-
DSN depletion is a simple method to remove selected transcripts from cDNA populations.Mol Biotechnol. 2009 Mar;41(3):247-53. doi: 10.1007/s12033-008-9131-y. Epub 2009 Jan 6. Mol Biotechnol. 2009. PMID: 19127453
-
Normalization of full-length enriched cDNA.Mol Biosyst. 2008 Mar;4(3):205-12. doi: 10.1039/b715110c. Epub 2008 Jan 8. Mol Biosyst. 2008. PMID: 18437263
-
Duplex-specific nuclease-mediated bioanalysis.Trends Biotechnol. 2015 Mar;33(3):180-8. doi: 10.1016/j.tibtech.2014.12.008. Epub 2015 Jan 29. Trends Biotechnol. 2015. PMID: 25640199 Review.
-
Recent advances in duplex-specific nuclease-based signal amplification strategies for microRNA detection.Biosens Bioelectron. 2020 Oct 1;165:112449. doi: 10.1016/j.bios.2020.112449. Epub 2020 Jul 14. Biosens Bioelectron. 2020. PMID: 32745963 Review.
Cited by
-
A catalogue of putative unique transcripts from Douglas-fir (Pseudotsuga menziesii) based on 454 transcriptome sequencing of genetically diverse, drought stressed seedlings.BMC Genomics. 2012 Nov 28;13:673. doi: 10.1186/1471-2164-13-673. BMC Genomics. 2012. PMID: 23190494 Free PMC article.
-
Brainstem transcriptomic changes in male Wistar rats after acute stress, comparing the use of duplex specific nuclease (DSN).Sci Rep. 2024 Sep 19;14(1):21856. doi: 10.1038/s41598-024-73042-2. Sci Rep. 2024. PMID: 39300279 Free PMC article.
-
Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.Sci Rep. 2016 Aug 24;6:31602. doi: 10.1038/srep31602. Sci Rep. 2016. PMID: 27554526 Free PMC article.
-
Transcriptome characterization via 454 pyrosequencing of the annelid Pristina leidyi, an emerging model for studying the evolution of regeneration.BMC Genomics. 2012 Jun 29;13:287. doi: 10.1186/1471-2164-13-287. BMC Genomics. 2012. PMID: 22747785 Free PMC article.
-
Contrasted evolution of the vomeronasal receptor repertoires in mammals and squamate reptiles.Genome Biol Evol. 2013;5(2):389-401. doi: 10.1093/gbe/evt013. Genome Biol Evol. 2013. PMID: 23348039 Free PMC article.
References
-
- Kato S., Sekine,S., Oh,S., Kim,N., Umezawa,Y., Abe,N., Yokoyama-Kobayashi,M. and Aoki,T. (1994) Construction of a human full-length cDNA bank. Gene, 150, 243–250. - PubMed
-
- Maruyama K. and Sugano,S. (1994) Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene, 138, 171–174. - PubMed
-
- Carninci P., Kvam,C., Kitamura,A., Ohsumi,T., Okazaki,Y., Itoh,M., Kamiya,M., Shibata,K., Sasaki,N., Izawa,M. et al. (1996) High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics, 37, 327–336. - PubMed
-
- Suzuki Y., Yoshitomo-Nakagawa,K., Maruyama,K., Suyama,A. and Sugano,S. (1997) Construction and characterization of a full length-enriched and a 5′-end-enriched cDNA library. Gene, 200, 149–156. - PubMed
Publication types
MeSH terms
Substances
Associated data
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
- Actions
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources