Trimmer and Trimmer-Direct cDNA normalization kits

cat.# NK001; NK002

- Acceleration of transcriptome analysis, functional screening, 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

Evrogen offers cDNA normalization kits designed to generate full-length-enriched double stranded (ds) cDNA with equalized concentrations of different transcripts. Both total RNA and poly(A+) RNA can be used for cDNA preparation and subsequent normalization. Depending on your particular needs, you can select Trimmer or Trimmer-Direct kit wherein the first allows preparation of normalized cDNA ready for nondirectional cloning and the second – for directional cloning of cDNA library. Alternatively, DSN-normalized cDNA can be used for 454 sequencing.

Method overview

cDNA normalization using duplex-specific nuclease (DSN) is a highly efficient approach that can be applied for normalization of full-length-enriched cDNA (Zhulidov et al., 2004; 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.

	Schematic outline of DSN-normalization. Black lines represent abundant transcripts, grey line – rare transcripts. Rectangle represents adapter sequence and its complement. Enlarge scheme

DSN isolated from kamchatka crab exhibits a strong preference for ds DNA as a substrate, and is stable under elevated temperatures (Shagin et al., 2002). Maximal DSN activity is observed at 60-65°C, and about 25% activity is retained even after incubation at 70°C for 20 min. These properties allow the effective removal of ds DNA, while ss fraction remains intact.

Owing to DSN thermostability, DSN-based degradation of ds DNA is performed under conditions of cDNA renaturation that prevent the formation of secondary structures and non-specific hybridization involving adapter sequences within the ss cDNA fraction.

DSN-normalization includes a step involving amplification of the normalized ss cDNA fraction. To overcome the PCR tendency to amplify shorter DNA fragments more efficiently than longer fragments, regulation of the average length of complex PCR products by partial PCR suppression is used (Shagin et al., 1999). To attain the PCR suppression effect, the cDNA to be amplified should contain inverted terminal repeats.

Adapter sequences can be introduced into cDNA ends by various means, for example, adapter ligation or during cDNA synthesis. Depending on the flanking adapters used in cDNA synthesis, normalized cDNA can be cloned directionally or non-directionally. A size-separation procedure is recommended to remove short cDNA fragments before cloning.

In addition to amplified ds DNA normalization, mostly required when only total RNA is available as a starting material, poly(A+) RNA-first-strand cDNA intermediates generated during first-strand cDNA synthesis are normalized using this method (Zhulidov et al., 2004). In this case, inverted terminal repeats at the ends of first-strand cDNA are required for subsequent PCR amplification.

Detailed protocols of DSN-normalization modifications are available in the book Nucleic Acids Hybridization Modern Applications (Shcheglov et al., 2007).

Typical cDNA normalization result. (A) Agarose/EtBr gel-electrophoresis of non-normalized (1) and normalized (2) human cDNA samples; (B, C) 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); embr. - embryonic; sm.intest. - small intestine.