A simple method for obtaining cell‐specific cDNA from small numbers of growing root‐hair cells in Arabidopsis thaliana

A simple and rapid method for cloning specific cDNAs from mRNA populations derived solely from small numbers of root‐hair cells is described here. To identify genes expressed during the earliest visible stage of root‐hair cell development, cell contents were aspirated from small numbers of Arabidops...

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Bibliographic Details
Published in:Journal of experimental botany 2003-05, Vol.54 (386), p.1373-1378
Main Authors: Jones, Mark A., Grierson, Claire S.
Format: Article
Language:English
Subjects:
Arabidopsis - cytology
Arabidopsis - genetics
Arabidopsis - growth & development
Arabidopsis Proteins - genetics
Biological and medical sciences
CDNA libraries
Cell walls
Cloning, Molecular - methods
Complementary DNA
Diverse techniques
DNA, Complementary - chemistry
DNA, Complementary - genetics
Epidermal cells
Fundamental and applied biological sciences. Psychology
Gene Library
GTP-Binding Proteins - genetics
Guard cells
Hair cells
Key words: Cell aspiration
Libraries
Messenger RNA
Molecular and cellular biology
nested PCR
Plant cells
Plant Roots - cytology
Plant Roots - genetics
Plant Roots - growth & development
Polymerase chain reaction
RESEARCH PAPER
rho GTP-Binding Proteins - genetics
RNA, Messenger - genetics
RNA, Messenger - isolation & purification
root hairs
Sequence Analysis, DNA
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Description
Summary:A simple and rapid method for cloning specific cDNAs from mRNA populations derived solely from small numbers of root‐hair cells is described here. To identify genes expressed during the earliest visible stage of root‐hair cell development, cell contents were aspirated from small numbers of Arabidopsis root‐hair cells at or just before this stage. This material was used to make reusable solid‐phase oligo‐dT‐primed cDNA libraries. To demonstrate that the libraries contained high quality longer cDNAs, a fragment located 2.7 kb from the 3′ end of the cDNA of the single copy root‐hair expressed gene RHD3 was cloned using a nested PCR strategy. This technique was also used to obtain novel gene expression information by cloning the full‐length 0.85 kb cDNA of the Rop2 GTPase from this library. This approach offers a means of cloning larger cDNAs directly from small numbers of growing root‐hair cells and, potentially, other epidermal cell types.
ISSN:0022-0957
1460-2431
1460-2431
DOI:10.1093/jxb/erg157