Open AccessIn situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries.
Author(s) -
Erik E. Karrer,
James E. Lincoln,
Saskia A. Hogenhout,
A. B. Bennett,
Richard M. Bostock,
Belinda Martineau,
William J. Lucas,
David G. Gilchrist,
Danny Alexander
Publication year - 1995
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.92.9.3814
Subject(s) - complementary dna , biology , cdna library , messenger rna , reverse transcriptase , rna , population , cloning (programming) , microbiology and biotechnology , rna extraction , template , cell , genetics , gene , nanotechnology , demography , materials science , sociology , computer science , programming language
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.