Single-Cell qRT-PCR with Fluidigm

We proceeded as described in Valente et al. (2019) (link). Cells were sorted in RT-STA reaction mix from the CellsDirect One-Step qRT-PCR Kit (Life Technologies), reverse transcribed and specific target pre-amplified (20 cycles), according to the manufacturer’s procedures. Pre-amplified samples were diluted 5x with low EDTA TE buffer prior to qPCR analysis using 48.48 Dynamic Array IFCs and the BioMark TM HD System (Fluidigm). The same TaqMan gene expression assays (20x, Life Technologies) were individually diluted 1:1 with 2x assay loading reagent (Fluidigm). Pre-amplified samples were combined with TaqMan Universal Master Mix (Life Technologies) and 20x GE sample loading reagent (Fluidigm). Loading of the 48.48 Dynamic Array TM IFCs and qPCR cycling conditions followed the Fluidigm procedure for TaqMan gene expression assays.

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Darrigrand J.F., Valente M., Comai G., Martinez P., Petit M., Nishinakamura R., Osorio D.S., Renault G., Marchiol C., Ribes V, & Cadot B. (2020). Dullard-mediated Smad1/5/8 inhibition controls mouse cardiac neural crest cells condensation and outflow tract septation. eLife, 9, e50325.

Publication 2020

CellsDirect One-Step qRT-PCR Kit BioMark TM HD System 2x assay loading reagent TaqMan Universal Master Mix 20x GE sample loading reagent

Assays Buffer Cells Edta Gene expression procedure

Corresponding Organization :

Other organizations : Centre de Recherche en Myologie, Inserm, Sorbonne Université, Paris Cardiovascular Research Center, Institut Pasteur, Centre National de la Recherche Scientifique, Kumamoto University, i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Université Paris Cité, Institut Cochin, Institut Jacques Monod

Top 5 similar protocols

Variable analysis

independent variables

Cell sorting
RT-STA reaction mix
Reverse transcription
Specific target pre-amplification (20 cycles)
Dilution of pre-amplified samples
TaqMan gene expression assays
Combination of pre-amplified samples with TaqMan Universal Master Mix and 20x GE sample loading reagent
Loading of the 48.48 Dynamic Array TM IFCs and qPCR cycling conditions

dependent variables

Gene expression measurement using the BioMark TM HD System (Fluidigm)

control variables

Manufacturer's procedures for CellsDirect One-Step qRT-PCR Kit (Life Technologies)
Fluidigm procedure for TaqMan gene expression assays

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