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. 2000 Oct;10(10):1617-30.
doi: 10.1101/gr.145100.

Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes

P Carninci  1 , Y Shibata, N Hayatsu, Y Sugahara, K Shibata, M Itoh, H Konno, Y Okazaki, M Muramatsu, Y Hayashizaki
Affiliations

Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes

P Carninci et al. Genome Res. 2000 Oct.

Abstract

In the effort to prepare the mouse full-length cDNA encyclopedia, we previously developed several techniques to prepare and select full-length cDNAs. To increase the number of different cDNAs, we introduce here a strategy to prepare normalized and subtracted cDNA libraries in a single step. The method is based on hybridization of the first-strand, full-length cDNA with several RNA drivers, including starting mRNA as the normalizing driver and run-off transcripts from minilibraries containing highly expressed genes, rearrayed clones, and previously sequenced cDNAs as subtracting drivers. Our method keeps the proportion of full-length cDNAs in the subtracted/normalized library high. Moreover, our method dramatically enhances the discovery of new genes as compared to results obtained by using standard, full-length cDNA libraries. This procedure can be extended to the preparation of full-length cDNA encyclopedias from other organisms.

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Figures

Figure 1
Figure 1
Schematic diagram of the normalized-subtracted cDNA preparation protocol. (A) general scheme for preparing full-length single-strand cDNA; (B) representation of various populations of tester cDNAs; (C) normalizing driver (cellular mRNA) and subtracting drivers (run-off transcripts); (D) hybridization; (E) rare/new cDNAs are used for second-strand cDNA preparation (normalized/subtracted cDNA library); (F) abundant cDNAs/unwanted cDNAs are removed and may be used for the preparation of minilibraries to implement subtraction.
Figure 2
Figure 2
Visualization of removal of highly abundant full-length cDNAs. Left, second-strand cDNA prepared with control pancreas cDNA; right, cDNA prepared with an aliquot of the same pancreas cDNA after normalization/subtraction. Highly abundant cDNAs are indicated with an arrow and are removed in the normalized/subtracted cDNAs.
Figure 3
Figure 3
Plaque hybridization of replicas containing control lung cDNA library (left) or a normalized lung cDNA library (right). In the right panel (normalized), an arrow indicates the plaque we have counted.
Figure 4
Figure 4
Sequencing redundancy (or the decrease in new gene discovery) increases sharply in standard cDNA libraries (-000 libraries), but in normalized/subtracted full-length cDNA libraries (-100 libraries), redundancy increases much more slowly. New genes (%) are referred as singleton (%) within a given cDNA library.
See this image and copyright information in PMC

Comment in

  • Of mice, men, and the genome.
    Margolin J. Margolin J. Genome Res. 2000 Oct;10(10):1431-2. doi: 10.1101/gr.162800. Genome Res. 2000. PMID: 11042142 No abstract available.

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