Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 301629
Title In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries
Author(s) Karrer, E.E.; Lincoln, J.E.; Hogenhout, S.A.; Bennett, A.B.; Bostock, R.M.; Martineau, B.; Lucas, W.J.; Gilchrist, D.G.; Alexander, D.
Source Proceedings of the National Academy of Sciences of the United States of America 92 (1995)9. - ISSN 0027-8424 - p. 3814 - 3818.
DOI https://doi.org/10.1073/pnas.92.9.3814
Department(s) Research Institute for Plant Protection
Publication type Refereed Article in a scientific journal
Publication year 1995
Abstract A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)-linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (< or = 0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.
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