Abstract
BackgroundThe incidence of false positives is a potential problem in single-cell PCR experiments. This paper describes an optimized protocol for single-cell qPCR measurements in primary pituitary cell cultures following patch-clamp recordings. Two different cell harvesting methods were assessed using both the GH4 prolactin producing cell line from rat, and primary cell culture from fish pituitaries.ResultsHarvesting whole cells followed by cell lysis and qPCR performed satisfactory on the GH4 cell line. However, harvesting of whole cells from primary pituitary cultures regularly produced false positives, probably due to RNA leakage from cells ruptured during the dispersion of the pituitary cells. To reduce RNA contamination affecting the results, we optimized the conditions by harvesting only the cytosol through a patch pipette, subsequent to electrophysiological experiments. Two important factors proved crucial for reliable harvesting. First, silanizing the patch pipette glass prevented foreign extracellular RNA from attaching to charged residues on the glass surface. Second, substituting the commonly used perforating antibiotic amphotericin B with β-escin allowed efficient cytosol harvest without loosing the giga seal. Importantly, the two harvesting protocols revealed no difference in RNA isolation efficiency.ConclusionDepending on the cell type and preparation, validation of the harvesting technique is extremely important as contaminations may give false positives. Here we present an optimized protocol allowing secure harvesting of RNA from single cells in primary pituitary cell culture following perforated whole cell patch clamp experiments.
Highlights
The incidence of false positives is a potential problem in single-cell PCR experiments
Using the patch clamp technique, we investigate the electrophysiological properties of single pituitary cells in culture [1], our main objective being to study the differences in electrical properties of follicle-stimulating hormone (FSH)- and luteinizing hormone (LH)-producing cells in teleost fish
The specificity of each qPCR primer pair was verified by first using a melting curve analysis followed by gel electrophoresis (Figure 2 for rat PRL and figure 3 for cod FSHb, LHb, and elongation factor 1a (EF1a))
Summary
The incidence of false positives is a potential problem in single-cell PCR experiments. Fan and Palade [12] and Sarantopoulos et al [13] successfully tested a more hydrophilic perforating agent, a saponin derived from the horse chestnut tree, called b-escin to reduce Ca2+ current rundown in rat neurons. They concluded that b-escin improves the giga seal formation and gives lower access resistance compared to traditional pore-forming antibiotics. The present study describes a protocol that reduces the risk of RNA contamination producing false positives, and at the same time facilitates the difficult transition from a perforated patch to a complete hole in the membrane necessary for cytosol harvesting
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