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cDNA Normalization

Evrogen´s Duplex Specific Nuclease Based TRIMMER Technology
Fast and reliable way to equalize abundance of different transcripts in a cDNA population and prepare full-length-enriched normalized cDNA pools/libraries for EST analysis and rare gene discovery

• Rapid and reliable way to remove repeated transcripts from cDNA library
• Equalization of full-length-enriched cDNA before library cloning
• Simple procedure, no physical separation steps
• Fully compatible with Illumina / Solexa, ABI / SOLiD and Roche / 454 sequencing protocols

In an eukaryotic cell, the mRNA population constitutes approximately 1% of total RNA with the number of transcripts varying from several thousand to several tens of thousands. Normally, the high abundance transcripts (several thousand mRNA copies per cell) of as few as 5-10 genes account for 20% of the cellular mRNA. The intermediate abundance transcripts (several hundred copies per cell) of 500-2000 genes constitute about 40-60% of the cellular mRNA. The remaining 20-40% of mRNA is represented by rare transcripts (from one to several dozen mRNA copies per cell) [Alberts et al., 1994]. Such an enormous difference in abundance complicates large-scale transcriptome analysis, which results in recurrent sequencing of more abundant cDNAs.

cDNA normalization decreases the prevalence of high abundance transcripts and equalizes transcript concentrations in a cDNA sample, thereby dramatically increasing the efficiency of sequencing and rare gene discovery.

The Trimmer-2 Kit is designed to normalize amplified full-length-enriched cDNA prepared using Evrogen Mint cDNA synthesis kits. The resulting cDNA contains equalized abundance of different transcripts and can be used for construction of cDNA libraries and for direct sequencing, including high-throughput sequencing on the next generation sequencing platforms (Roche/454, ABI/SOLiD or Illumina/Solexa). The kit also includes special adapters allowing use of Clontech SMART-based kits for construction of cDNA intended for Trimmer-2 normalization.

Starting Materials
- Mint-2 cDNA Synthesis Kit (Cat.# SK005-EV) recommended
- SMARTer PCR cDNA synthesis kit (Clontech Cat.# 634925 or 634926)*
- SMART cDNA Library Construction kit (Clontech Cat.# 634901)*
- SMART PCR cDNA synthesis kit (Clontech Cat.# 634902)
- Creator SMART cDNA Library Construction kit (Clontech Cat.# 634903)*.

*Please follow instructions provided in the Trimmer-2 Kit user manual for cDNA synthesis using SMART kits. Please note that CDS adapters included in the Trimmer-2 Kit must be used for cDNA preparation instead of CDS primers included in SMART kits.

Method Overview
cDNA normalization using duplex-specific nuclease (DSN) from kamchatka crab is a highly efficient approach that can be applied for normalization of full-length-enriched cDNA (Zhulidov et al., 2004; Zhulidov et al., 2005). Both total and poly(A)+ RNA can be used as a starting material. DSN-normalization has been successfully applied to various animal and plant models (see Bogdanova et al., 2008 for review). The flexibility of this normalization procedure allows simple modifications for various purposes.
DSN-normalization is performed prior to library cloning, and involves the denaturation of ds cDNA flanked with known adapters, its subsequent renaturation, and enzymatic degradation of the ds DNA fraction (formed by abundant transcripts) by DSN. The equalized ss cDNA fraction remains intact, and is amplified by PCR.
For details, see link below.

Image
Typical cDNA normalization result.
(A) Agarose/EtBr gel-electrophoresis of non-normalized (1) and normalized (2) human cDNA samples. (B) concentration of abundant transcripts in these samples revealed by Virtual Northern blot. GPDH – glyceraldehyde-3-phosphate dehydrogenase; UBC – ubiquitin C; M, 1-kb DNA size markers (SibEnzyme).
(C) Normalization significantly increases gene discovery rate of cDNA library. Transcript distribution in standard and normalized cDNA libraries from Aplysia neurons: yellow – all sequences, blue – unique sequences; green – non-unique sequences.

References
Alberts B et al. Molecular biology of the cell. 3rd ed., Garland Publishing, New York. 1994.
Bogdanova EA et al. Normalization of full-length enriched cDNA. Mol Biosyst. 2008; 4 (3):205-12. / pmid: 18437263
Shagin DA et al. Regulation of average length of complex PCR product. Nucleic Acids Res. 1999; 27 (18):e23. / pmid: 10471753
Shagin DA et al. A novel method for SNP detection using a new duplex-specific nuclease from crab hepatopancreas. Genome Res. 2002; 12 (12):1935-42. / pmid: 12466298
Shcheglov A et al. Normalization of cDNA Libraries. In: Nucleic Acids Hybridization: Modern Applications. Buzdin, Anton; Lukyanov, Sergey (Eds.) ISBN: 978-1-4020-6039-7. 2007; 97-124.
Zhulidov PA et al. A method for the preparation of normalized cDNA libraries enriched with full-length sequences. Bioorg Khim. 2005; 31 (2):186-94. pmid: 15889793
Zhulidov PA et al. Simple cDNA normalization using kamchatka crab duplex-specific nuclease. Nucleic Acids Res. 2004; 32 (3):e37. / pmid: 14973331

Duplex-Specific Nuclease (DSN) from Kamchatka crab is also available as a seperate product, when you like to use your own protocols.

Notice to Purchaser
Evrogen DSN Products: Evrogen Duplex-Specific Nuclease Products are available to Purchasers for non-commercial non-for-profit research use.
Related Links

MINT Full Length Enriched cDNA Synthesis Kits
Detailed Info about DSN Normalization Technology
Duplex Specific Nuclease


PDF-Downloads - Will open in new browser window

User Manual Trimmer-2 cDNA Normalization Kit


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