qRT-PCR RNA Isolation and Analysis

RNA isolation was performed with NucleoSpin RNA/Protein kits from Macherey-Nagel according to the manufactures instructions including on column DNase treatment. The RNA concentration and quality was measured with spectroscopy (Nanodrop, Thermo Fisher Scientific) and capillary electrophoresis (Agilent 2100 Bioanalyzer). Approximately 1 µg total RNA was reverse transcribed using the Multi-Scribe RT kit (Applied Biosystems, Carlsbad, CA) with random hexamers. Reactions were performed in triplicate using SYBR Green I master mix (Applied Biosystems, Carlsbad, CA). Normalization and error propagation were calculated as described [116] (link). Relative quantities were normalized to beta-actin. Sequences of qPCR primer pairs are provided in S8 Table.

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Isensee J., Wenzel C., Buschow R., Weissmann R., Kuss A.W, & Hucho T. (2014). Subgroup-Elimination Transcriptomics Identifies Signaling Proteins that Define Subclasses of TRPV1-Positive Neurons and a Novel Paracrine Circuit. PLoS ONE, 9(12), e115731.

Publication 2014

NucleoSpin RNA/Protein kits Nanodrop Agilent 2100 Bioanalyzer Multi-Scribe RT kit SYBR Green I master mix

Beta actin Capillary electrophoresis Dnase Isolation Primer Protein Spectroscopy Sybr green i

Corresponding Organization : Universität Greifswald

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Variable analysis

independent variables

RNA isolation was performed with NucleoSpin RNA/Protein kits from Macherey-Nagel according to the manufactures instructions including on column DNase treatment.

dependent variables

The RNA concentration and quality was measured with spectroscopy (Nanodrop, Thermo Fisher Scientific) and capillary electrophoresis (Agilent 2100 Bioanalyzer).
Relative quantities were normalized to beta-actin.

control variables

Approximately 1 µg total RNA was reverse transcribed using the Multi-Scribe RT kit (Applied Biosystems, Carlsbad, CA) with random hexamers.
Reactions were performed in triplicate using SYBR Green I master mix (Applied Biosystems, Carlsbad, CA).

positive controls

None specified.

negative controls

None specified.

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