First patch, then catch: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen protoplasts1
- 18 January 2002
- journal article
- Published by Wiley in FEBS Letters
- Vol. 512 (1-3) , 152-156
- https://doi.org/10.1016/s0014-5793(02)02246-9
Abstract
Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H+ ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all ‘patch–catch’ experiments and could be detected even in 2560-fold dilutions of the pollen contents. H+ ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT-PCR experiments, a random non-reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts.Keywords
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