title: Digital PCR to determine the number of transcripts from single neurons after patch-clamp recording.
abstract: Whole-cell patch-clamp recording enables detection of electrophysiological signals from single neurons as well as harvesting of perisomatic RNA through the patch pipette for subsequent gene expression analysis. Amplification and profiling of RNA with traditional quantitative real-time PCR (qRT-PCR) do not provide exact quantitation due to experimental variation caused by the limited amount of nucleic acid in a single cell. Here we describe a protocol for quantifying mRNA or miRNA expression in individual neurons after patch-clamp recording using high-density nanocapillary digital PCR (dPCR). Expression of a known cell-type dependent marker gene (gabrd), as well as oxidative-stress related induction of hspb1 and hmox1 expression, was quantified in individual neurogliaform and pyramidal cells, respectively. The miRNA mir-132, which plays a role in neurodevelopment, was found to be equally expressed in three different types of neurons. The accuracy and sensitivity of this method were further validated using synthetic spike-in templates and by detecting genes with very low levels of expression.
authors.affiliation: Avidin Ltd, Szeged, Hungary.
authors.forename: Nóra
authors.initials: N
authors.lastname: Faragó
authors.forename: Ágnes K
authors.initials: ÁK
authors.lastname: Kocsis
authors.forename: Sándor
authors.initials: S
authors.lastname: Lovas
authors.forename: Gábor
authors.initials: G
authors.lastname: Molnár
authors.forename: Eszter
authors.initials: E
authors.lastname: Boldog
authors.forename: Márton
authors.initials: M
authors.lastname: Rózsa
authors.forename: Viktor
authors.initials: V
authors.lastname: Szemenyei
authors.forename: Enikő
authors.initials: E
authors.lastname: Vámos
authors.forename: Lajos I
authors.initials: LI
authors.lastname: Nagy
authors.forename: Gábor
authors.initials: G
authors.lastname: Tamás
authors.forename: László G
authors.initials: LG
authors.lastname: Puskás
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods; D008297:Male; D035683:MicroRNAs / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D059010:Single-Cell Analysis / Q000379:methods*; D013003:Somatosensory Cortex / Q000166:cytology
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger