IN-SITU ISOLATION OF MESSENGER-RNA FROM INDIVIDUAL PLANT-CELLS - CREATION OF CELL-SPECIFIC CDNA LIBRARIES

A method for isolating and cloning mRNA populations from individual ce lls in living, intact plant tissues is described, The contents of indi vidual cells were aspirated into micropipette tips filled with RNA ext raction buffer. The mRNA from these cells was purified by binding to o ligo(dT)-linked magnetic beads and amplified on the beads using revers e transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf e pidermal and guard cell mRNA preparations. In testing the reproducibil ity of the method, we discovered an inherent limitation of PCR amplifi cation from small amounts of any complex template. This phenomenon, wh ich we have termed the ''Monte Carlo'' effect, is created by small and random differences in amplification efficiency between individual tem plates in an amplifying cDNA population. The Monte Carlo effect is dep endent upon template concentration: the lower the abundance of any tem plate, the less likely its true abundance will be reflected in the amp lified library. Quantitative assessment of the Monte Carlo effect reve aled that only rare mRNAs (less than or equal to 0.04% of polyadenylyl ated mRNA) exhibited significant variation in amplification at the sin gle-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.