Format

Send to

Choose Destination
See comment in PubMed Commons below
Biotechniques. 2013 Jun;54(6):327-36. doi: 10.2144/000114029.

Digital PCR to determine the number of transcripts from single neurons after patch-clamp recording.

Author information

1
Avidin Ltd, Szeged, Hungary.

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.

PMID:
23750542
DOI:
10.2144/000114029
[Indexed for MEDLINE]
Free full text
PubMed Commons home

PubMed Commons

0 comments

    Supplemental Content

    Full text links

    Icon for Informa Healthcare, USA
    Loading ...
    Support Center